SEC Info  
    Home      Search      My Interests      Help      Sign In      Please Sign In

Harvard Bioscience Inc. – ‘10-K’ for 12/31/23 – ‘EX-101.DEF’

On:  Thursday, 3/7/24, at 5:00pm ET   ·   For:  12/31/23   ·   Accession #:  1171843-24-1239   ·   File #:  1-33957

Previous ‘10-K’:  ‘10-K’ on 3/9/23 for 12/31/22   ·   Latest ‘10-K’:  This Filing   ·   20 References:   

Find Words in Filings emoji
 
  in    Show  and   Hints

  As Of               Filer                 Filing    For·On·As Docs:Size             Issuer                      Filing Agent

 3/07/24  Harvard Bioscience Inc.           10-K       12/31/23  113:9.7M                                   Globenewswire Inc./FA

Annual Report   —   Form 10-K   —   SEA’34

Filing Table of Contents

Document/Exhibit                   Description                      Pages   Size 

 1: 10-K        Annual Report                                       HTML   1.71M 
 2: EX-21.1     Subsidiaries List                                   HTML     31K 
 3: EX-23.1     Consent of Expert or Counsel                        HTML     30K 
 8: EX-97       Clawback Policy re: Recovery of Erroneously         HTML     38K 
                Awarded Compensation                                             
 4: EX-31.1     Certification -- §302 - SOA'02                      HTML     35K 
 5: EX-31.2     Certification -- §302 - SOA'02                      HTML     35K 
 6: EX-32.1     Certification -- §906 - SOA'02                      HTML     31K 
 7: EX-32.2     Certification -- §906 - SOA'02                      HTML     31K 
14: R1          Document And Entity Information                     HTML    100K 
15: R2          Consolidated Balance Sheets                         HTML    137K 
16: R3          Consolidated Balance Sheets (Parentheticals)        HTML     43K 
17: R4          Consolidated Statements of Operations               HTML    102K 
18: R5          Consolidated Statements of Comprehensive Loss       HTML     60K 
19: R6          Consolidated Statements of Comprehensive Loss       HTML     33K 
                (Parentheticals)                                                 
20: R7          Consolidated Statements of Stockholders' Equity     HTML     89K 
21: R8          Consolidated Statements of Cash Flows               HTML    120K 
22: R9          Insider Trading Arrangements                        HTML     49K 
23: R10         Note 1 - Organization                               HTML     34K 
24: R11         Note 2 - Summary of Significant Accounting          HTML    113K 
                Policies                                                         
25: R12         Note 3 - Accumulated Other Comprehensive Loss       HTML     52K 
26: R13         Note 4 - Goodwill and Intangible Assets             HTML     78K 
27: R14         Note 5 - Balance Sheet Information                  HTML     73K 
28: R15         Note 6 - Restructuring and Other Exit Costs         HTML     58K 
29: R16         Note 7 - Employee Benefit Plans                     HTML    111K 
30: R17         Note 8 - Leases                                     HTML     68K 
31: R18         Note 9 - Long-term Debt                             HTML     56K 
32: R19         Note 10 - Derivatives                               HTML     43K 
33: R20         Note 11 - Fair Value Measurements                   HTML     43K 
34: R21         Note 12 - Stock-Based Compensation                  HTML    100K 
35: R22         Note 13 - Revenues                                  HTML     92K 
36: R23         Note 14 - Income Tax                                HTML    130K 
37: R24         Note 15 - Commitments and Contingent Liabilities    HTML     37K 
38: R25         Note 16 - Litigation Settlement                     HTML     35K 
39: R26         Note 17 - Product Line Disposition                  HTML     34K 
40: R27         Note 18 - Government Assistance                     HTML     36K 
41: R28         Significant Accounting Policies (Policies)          HTML    172K 
42: R29         Note 2 - Summary of Significant Accounting          HTML     50K 
                Policies (Tables)                                                
43: R30         Note 3 - Accumulated Other Comprehensive Loss       HTML     51K 
                (Tables)                                                         
44: R31         Note 4 - Goodwill and Intangible Assets (Tables)    HTML     82K 
45: R32         Note 5 - Balance Sheet Information (Tables)         HTML     77K 
46: R33         Note 6 - Restructuring and Other Exit Costs         HTML     55K 
                (Tables)                                                         
47: R34         Note 7 - Employee Benefit Plans (Tables)            HTML    114K 
48: R35         Note 8 - Leases (Tables)                            HTML     72K 
49: R36         Note 9 - Long-term Debt (Tables)                    HTML     49K 
50: R37         Note 10 - Derivatives (Tables)                      HTML     44K 
51: R38         Note 11 - Fair Value Measurements (Tables)          HTML     40K 
52: R39         Note 12 - Stock-Based Compensation (Tables)         HTML     99K 
53: R40         Note 13 - Revenues (Tables)                         HTML     94K 
54: R41         Note 14 - Income Tax (Tables)                       HTML    129K 
55: R42         Note 2 - Summary of Significant Accounting          HTML     77K 
                Policies (Details Textual)                                       
56: R43         Note 2 - Summary of Significant Accounting          HTML     39K 
                Policies - Property, Plant and Equipment (Details)               
57: R44         Note 2 - Summary of Significant Accounting          HTML     64K 
                Policies - Schedule of Basic and Diluted Earnings                
                Per Share (Details)                                              
58: R45         Note 3 - Accumulated Other Comprehensive Loss -     HTML     49K 
                Changes in Each Component of Other Comprehensive                 
                Loss, Net of Tax (Details)                                       
59: R46         Note 4 - Goodwill and Intangible Assets (Details    HTML     35K 
                Textual)                                                         
60: R47         Note 4 - Goodwill and Intangible Assets - Change    HTML     35K 
                in Carrying Amount of Goodwill (Details)                         
61: R48         Note 4 - Goodwill and Intangible Assets -           HTML     52K 
                Intangible Assets (Details)                                      
62: R49         Note 4 - Goodwill and Intangible Assets - Future    HTML     45K 
                Amortization Expense of Intangible Assets                        
                (Details)                                                        
63: R50         Note 5 - Balance Sheet Information (Details         HTML     33K 
                Textual)                                                         
64: R51         Note 5 - Balance Sheet Information - Inventories    HTML     38K 
                (Details)                                                        
65: R52         Note 5 - Balance Sheet Information - Property,      HTML     46K 
                Plant and Equipment (Details)                                    
66: R53         Note 5 - Balance Sheet Information - Other Current  HTML     42K 
                Liabilities (Details)                                            
67: R54         Note 5 - Balance Sheet Information - Long-lived     HTML     35K 
                Assets by Geographic Area (Details)                              
68: R55         Note 6 - Restructuring and Other Exit Costs -       HTML     48K 
                Restructuring and Other Exit Charges (Details)                   
69: R56         Note 7 - Employee Benefit Plans (Details Textual)   HTML     46K 
70: R57         Note 7 - Employee Benefit Plans - Defined Benefit   HTML     41K 
                Pension Expense (Details)                                        
71: R58         Note 7 - Employee Benefit Plans - Change in Fair    HTML     44K 
                Value of Plan Assets (Details)                                   
72: R59         Note 7 - Employee Benefit Plans - Change in         HTML     52K 
                Benefit Obligations (Details)                                    
73: R60         Note 7 - Employee Benefit Plans - Recognized in     HTML     33K 
                Consolidated Balance Sheets (Details)                            
74: R61         Note 7 - Employee Benefit Plans - Weighted Average  HTML     33K 
                Assumptions (Details)                                            
75: R62         Note 7 - Employee Benefit Plans - Fair Value and    HTML     45K 
                Asset Allocations of Pension Benefits (Details)                  
76: R63         Note 7 - Employee Benefit Plans - Fair Value of     HTML     38K 
                Plan Assets By Fair Value Hierarchy (Details)                    
77: R64         Note 8 - Leases - Lease Expense (Details)           HTML     37K 
78: R65         Note 8 - Leases - Supplemental Balance Sheet        HTML     41K 
                Information Related to Operating Leases (Details)                
79: R66         Note 8 - Leases - Supplemental Cash Flow            HTML     33K 
                Information Related to Operating Leases (Details)                
80: R67         Note 8 - Leases - Future Minimum Lease Payments     HTML     48K 
                (Details)                                                        
81: R68         Note 9 - Long-term Debt (Details Textual)           HTML     71K 
82: R69         Note 9 - Long-term Debt - Breakdown of Borrowings   HTML     47K 
                (Details)                                                        
83: R70         Note 9 - Long Term Debt - Debt Maturity (Details)   HTML     37K 
84: R71         Note 10 - Derivatives (Details Textual)             HTML     36K 
85: R72         Note 10 - Derivatives - Derivative Instruments      HTML     34K 
                (Details)                                                        
86: R73         Note 10 - Derivatives - The Effect of Derivatives   HTML     34K 
                Designated as Cash Flow Hedging Instruments                      
                (Details)                                                        
87: R74         Note 11 - Fair Value Measurements - Assets and      HTML     50K 
                Liabilities Measured on a Recurring Basis                        
                (Details)                                                        
88: R75         Note 12 - Stock-Based Compensation (Details         HTML     57K 
                Textual)                                                         
89: R76         Note 12 - Stock-Based Compensation - Stock-based    HTML     39K 
                Compensation Expense (Details)                                   
90: R77         Note 12 - Stock-Based Compensation - Black Scholes  HTML     42K 
                Assumptions (Details)                                            
91: R78         Note 12 - Stock-Based Compensation - Activity of    HTML     71K 
                Restricted Stock and Restricted Stock Units                      
                (Details)                                                        
92: R79         Note 12 - Stock-Based Compensation - Stock-Based    HTML     50K 
                Payment Awards (Details)                                         
93: R80         Note 13 - Revenues (Details Textual)                HTML     31K 
94: R81         Note 13 - Revenues - Disaggregation of Revenue      HTML     41K 
                (Details)                                                        
95: R82         Note 13 - Revenues - Revenue by Geographic          HTML     40K 
                Destination (Details)                                            
96: R83         Note 13 - Revenues - Deferred Revenue (Details)     HTML     38K 
97: R84         Note 13 - Revenues - Allowance for Doubtful         HTML     36K 
                Accounts (Details)                                               
98: R85         Note 13 - Revenues - Warranties (Details)           HTML     35K 
99: R86         Note 14 - Income Tax (Details Textual)              HTML     61K 
100: R87         Note 14 - Income Tax - Income Tax Expense           HTML     48K  
                (Details)                                                        
101: R88         Note 14 - Income Tax - Income Tax Rate              HTML     59K  
                Reconciliation (Details)                                         
102: R89         Note 14 - Income Tax - Pre-tax Income From          HTML     38K  
                Continuing Operations (Details)                                  
103: R90         Note 14 - Income Tax - Deferred Tax Assets and      HTML     74K  
                Liabilities (Details)                                            
104: R91         Note 14 - Income Tax - Unrecognized Tax Benefits    HTML     40K  
                (Details)                                                        
105: R92         Note 15 - Commitments and Contingent Liabilities    HTML     33K  
                (Details Textual)                                                
106: R93         Note 16 - Litigation Settlement (Details Textual)   HTML     53K  
107: R94         Note 17 - Product Line Disposition (Details         HTML     40K  
                Textual)                                                         
108: R95         Note 18 - Government Assistance (Details Textual)   HTML     35K  
110: XML         IDEA XML File -- Filing Summary                      XML    211K  
113: XML         XBRL Instance -- hboi20231231_10k_htm                XML   2.15M  
109: EXCEL       IDEA Workbook of Financial Report Info              XLSX    163K  
10: EX-101.CAL  XBRL Calculations -- hbio-20231231_cal               XML    212K 
11: EX-101.DEF  XBRL Definitions -- hbio-20231231_def                XML   1.55M 
12: EX-101.LAB  XBRL Labels -- hbio-20231231_lab                     XML   1.34M 
13: EX-101.PRE  XBRL Presentations -- hbio-20231231_pre              XML   1.64M 
 9: EX-101.SCH  XBRL Schema -- hbio-20231231                         XSD    249K 
111: JSON        XBRL Instance as JSON Data -- MetaLinks              591±   953K  
112: ZIP         XBRL Zipped Folder -- 0001171843-24-001239-xbrl      Zip    336K  


‘EX-101.DEF’   —   XBRL Definitions — hbio-20231231_def


This Exhibit is an XBRL File.


                                                                                                                                                                                
<?xml version="1.0" standalone="no" encoding="windows-1252"?>
<!-- Generated by ThunderDome XBRL - 09:46PM UTC 2024-03-07 -->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
<link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple"/>
<link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple"/>
<link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple"/>
<link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple"/>
<link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple"/>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements" xlink:href="hbio-20231231.xsd#statement-note-11-fair-value-measurements" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueDisclosuresTextBlock" xlink:label="us-gaap_FairValueDisclosuresTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_FairValueDisclosuresTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases" xlink:href="hbio-20231231.xsd#statement-note-8-leases" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeasesTextBlock" xlink:label="us-gaap_LesseeOperatingLeasesTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_LesseeOperatingLeasesTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs" xlink:href="hbio-20231231.xsd#statement-note-6-restructuring-and-other-exit-costs" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestructuringAndRelatedActivitiesDisclosureTextBlock" xlink:label="us-gaap_RestructuringAndRelatedActivitiesDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_RestructuringAndRelatedActivitiesDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss" xlink:href="hbio-20231231.xsd#statement-note-3-accumulated-other-comprehensive-loss" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ComprehensiveIncomeNoteTextBlock" xlink:label="us-gaap_ComprehensiveIncomeNoteTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_ComprehensiveIncomeNoteTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-1-organization" xlink:href="hbio-20231231.xsd#statement-note-1-organization" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-1-organization" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:label="us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-document-and-entity-information" xlink:href="hbio-20231231.xsd#statement-document-and-entity-information" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-document-and-entity-information" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorFirmId" xlink:label="dei_AuditorFirmId" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorLocation" xlink:label="dei_AuditorLocation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorName" xlink:label="dei_AuditorName" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentAnnualReport" xlink:label="dei_DocumentAnnualReport" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFinStmtErrorCorrectionFlag" xlink:label="dei_DocumentFinStmtErrorCorrectionFlag" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentInformationLineItems" xlink:label="dei_DocumentInformationLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentInformationTable" xlink:label="dei_DocumentInformationTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentType" xlink:label="dei_DocumentType" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressStateOrProvince" xlink:label="dei_EntityAddressStateOrProvince" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityPublicFloat" xlink:label="dei_EntityPublicFloat" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityVoluntaryFilers" xlink:label="dei_EntityVoluntaryFilers" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityWellKnownSeasonedIssuer" xlink:label="dei_EntityWellKnownSeasonedIssuer" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_IcfrAuditorAttestationFlag" xlink:label="dei_IcfrAuditorAttestationFlag" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="dei_DocumentInformationTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentInformationTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCentralIndexKey" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityRegistrantName" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AmendmentFlag" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CurrentFiscalYearEndDate" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalYearFocus" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentType" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentAnnualReport" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentPeriodEndDate" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentTransitionReport" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFileNumber" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityTaxIdentificationNumber" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressAddressLine1" xlink:type="arc"/>
<link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCityOrTown" xlink:type="arc"/>
<link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressStateOrProvince" xlink:type="arc"/>
<link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressPostalZipCode" xlink:type="arc"/>
<link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CityAreaCode" xlink:type="arc"/>
<link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_LocalPhoneNumber" xlink:type="arc"/>
<link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_Security12bTitle" xlink:type="arc"/>
<link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_TradingSymbol" xlink:type="arc"/>
<link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_SecurityExchangeName" xlink:type="arc"/>
<link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityWellKnownSeasonedIssuer" xlink:type="arc"/>
<link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityVoluntaryFilers" xlink:type="arc"/>
<link:definitionArc order="25" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCurrentReportingStatus" xlink:type="arc"/>
<link:definitionArc order="26" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityInteractiveDataCurrent" xlink:type="arc"/>
<link:definitionArc order="27" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFilerCategory" xlink:type="arc"/>
<link:definitionArc order="28" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntitySmallBusiness" xlink:type="arc"/>
<link:definitionArc order="29" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityEmergingGrowthCompany" xlink:type="arc"/>
<link:definitionArc order="30" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_IcfrAuditorAttestationFlag" xlink:type="arc"/>
<link:definitionArc order="31" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFinStmtErrorCorrectionFlag" xlink:type="arc"/>
<link:definitionArc order="32" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityShellCompany" xlink:type="arc"/>
<link:definitionArc order="33" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityPublicFloat" xlink:type="arc"/>
<link:definitionArc order="34" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" xlink:type="arc"/>
<link:definitionArc order="35" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorFirmId" xlink:type="arc"/>
<link:definitionArc order="36" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorName" xlink:type="arc"/>
<link:definitionArc order="37" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorLocation" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-balance-sheets-parentheticals" xlink:href="hbio-20231231.xsd#statement-consolidated-balance-sheets-parentheticals" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-balance-sheets-parentheticals" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockNoParValue" xlink:label="us-gaap_PreferredStockNoParValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockNoParValue" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-comprehensive-loss-parentheticals" xlink:href="hbio-20231231.xsd#statement-consolidated-statements-of-comprehensive-loss-parentheticals" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-comprehensive-loss-parentheticals" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossReclassificationAdjustmentFromAOCIPensionAndOtherPostretirementBenefitPlansForNetGainLossTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossReclassificationAdjustmentFromAOCIPensionAndOtherPostretirementBenefitPlansForNetGainLossTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossReclassificationAdjustmentFromAOCIPensionAndOtherPostretirementBenefitPlansForNetGainLossTax" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies" xlink:href="hbio-20231231.xsd#statement-note-2-summary-of-significant-accounting-policies" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementAdjustmentMember" xlink:label="srt_RestatementAdjustmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementAxis" xlink:label="srt_RestatementAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SignificantAccountingPoliciesTextBlock" xlink:label="us-gaap_SignificantAccountingPoliciesTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RestatementAxis" xlink:to="srt_RestatementDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RestatementAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RestatementDomain" xlink:to="srt_RestatementAdjustmentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RestatementAxis" xlink:to="srt_RestatementDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SignificantAccountingPoliciesTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GoodwillAndIntangibleAssetsDisclosureTextBlock" xlink:label="us-gaap_GoodwillAndIntangibleAssetsDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_GoodwillAndIntangibleAssetsDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SupplementalBalanceSheetDisclosuresTextBlock" xlink:label="us-gaap_SupplementalBalanceSheetDisclosuresTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SupplementalBalanceSheetDisclosuresTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PensionAndOtherPostretirementBenefitsDisclosureTextBlock" xlink:label="us-gaap_PensionAndOtherPostretirementBenefitsDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_PensionAndOtherPostretirementBenefitsDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt" xlink:href="hbio-20231231.xsd#statement-note-9-longterm-debt" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SecuredOvernightFinancingRateSofrMember" xlink:label="hbio_SecuredOvernightFinancingRateSofrMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SwinglineLoanFacilityMember" xlink:label="hbio_SwinglineLoanFacilityMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TermLoanMember" xlink:label="hbio_TermLoanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheABRLoanMember" xlink:label="hbio_TheABRLoanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheCreditAgreementMember" xlink:label="hbio_TheCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheLendersMember" xlink:label="hbio_TheLendersMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:label="hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BaseRateMember" xlink:label="us-gaap_BaseRateMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityAxis" xlink:label="us-gaap_CreditFacilityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LetterOfCreditMember" xlink:label="us-gaap_LetterOfCreditMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityAxis" xlink:label="us-gaap_LineOfCreditFacilityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtTextBlock" xlink:label="us-gaap_LongTermDebtTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevolvingCreditFacilityMember" xlink:label="us-gaap_RevolvingCreditFacilityMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LineOfCreditFacilityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LineOfCreditFacilityLenderDomain" xlink:to="hbio_TheLendersMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LongtermDebtTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="hbio_TermLoanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="hbio_TheCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_CreditFacilityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="us-gaap_RevolvingCreditFacilityMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="us-gaap_LetterOfCreditMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="hbio_SwinglineLoanFacilityMember" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VariableRateAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="hbio_SecuredOvernightFinancingRateSofrMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_BaseRateMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="hbio_TheABRLoanMember" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_LongTermDebtTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives" xlink:href="hbio-20231231.xsd#statement-note-10-derivatives" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheCreditAgreementMember" xlink:label="hbio_TheCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeInstrumentRiskAxis" xlink:label="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativesAndFairValueTextBlock" xlink:label="us-gaap_DerivativesAndFairValueTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestRateSwapMember" xlink:label="us-gaap_InterestRateSwapMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativeContractTypeDomain" xlink:to="us-gaap_InterestRateSwapMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="hbio_TheCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DerivativesAndFairValueTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EmployeeStockPurchasePlanMember" xlink:label="hbio_EmployeeStockPurchasePlanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TwentyTwentyOneIncentivePlanMember" xlink:label="hbio_TwentyTwentyOneIncentivePlanMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareholdersEquityAndShareBasedPaymentsTextBlock" xlink:label="us-gaap_ShareholdersEquityAndShareBasedPaymentsTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="hbio_TwentyTwentyOneIncentivePlanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="hbio_EmployeeStockPurchasePlanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_ShareholdersEquityAndShareBasedPaymentsTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues" xlink:href="hbio-20231231.xsd#statement-note-13-revenues" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerTextBlock" xlink:label="us-gaap_RevenueFromContractWithCustomerTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_RevenueFromContractWithCustomerTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_Expire2024Member" xlink:label="hbio_Expire2024Member" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ExpireBetween2024And2043Member" xlink:label="hbio_ExpireBetween2024And2043Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesAxis" xlink:label="srt_ConsolidatedEntitiesAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesDomain" xlink:label="srt_ConsolidatedEntitiesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesDomain" xlink:label="srt_ConsolidatedEntitiesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SubsidiariesMember" xlink:label="srt_SubsidiariesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ForeignCountryMember" xlink:label="us-gaap_ForeignCountryMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxDisclosureTextBlock" xlink:label="us-gaap_IncomeTaxDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonUsMember" xlink:label="us-gaap_NonUsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchMember" xlink:label="us-gaap_ResearchMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StateAndLocalJurisdictionMember" xlink:label="us-gaap_StateAndLocalJurisdictionMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAxis" xlink:label="us-gaap_TaxCreditCarryforwardAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodAxis" xlink:label="us-gaap_TaxPeriodAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodDomain" xlink:label="us-gaap_TaxPeriodDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodDomain" xlink:label="us-gaap_TaxPeriodDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ConsolidatedEntitiesAxis" xlink:to="srt_ConsolidatedEntitiesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxPeriodAxis" xlink:to="us-gaap_TaxPeriodDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_StateAndLocalJurisdictionMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxPeriodAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxPeriodDomain" xlink:to="hbio_ExpireBetween2024And2043Member" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxPeriodAxis" xlink:to="us-gaap_TaxPeriodDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_ForeignCountryMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxPeriodDomain" xlink:to="hbio_Expire2024Member" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxCreditCarryforwardAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxCreditCarryforwardNameDomain" xlink:to="us-gaap_ResearchMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_StatementGeographicalAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="us-gaap_NonUsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain-default" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ConsolidatedEntitiesAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ConsolidatedEntitiesDomain" xlink:to="srt_SubsidiariesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ConsolidatedEntitiesAxis" xlink:to="srt_ConsolidatedEntitiesDomain-default" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_IncomeTaxDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-15-commitments-and-contingent-liabilities" xlink:href="hbio-20231231.xsd#statement-note-15-commitments-and-contingent-liabilities" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-15-commitments-and-contingent-liabilities" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_IndemnificationAgreementsMember" xlink:label="hbio_IndemnificationAgreementsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_DirectorMember" xlink:label="srt_DirectorMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:label="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_TitleOfIndividualAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="srt_DirectorMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="hbio_IndemnificationAgreementsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-16-litigation-settlement" xlink:href="hbio-20231231.xsd#statement-note-16-litigation-settlement" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-16-litigation-settlement" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_CaseInSuffolkSuperiorCourtMember" xlink:label="hbio_CaseInSuffolkSuperiorCourtMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:label="hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseAxis" xlink:label="srt_LitigationCaseAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseTypeDomain" xlink:label="srt_LitigationCaseTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseTypeDomain" xlink:label="srt_LitigationCaseTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConvertiblePreferredStockMember" xlink:label="us-gaap_ConvertiblePreferredStockMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameAxis" xlink:label="us-gaap_InvestmentIssuerNameAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameDomain" xlink:label="us-gaap_InvestmentIssuerNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameDomain" xlink:label="us-gaap_InvestmentIssuerNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LegalMattersAndContingenciesTextBlock" xlink:label="us-gaap_LegalMattersAndContingenciesTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_LitigationCaseAxis" xlink:to="srt_LitigationCaseTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_InvestmentIssuerNameAxis" xlink:to="us-gaap_InvestmentIssuerNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_LitigationCaseAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_LitigationCaseTypeDomain" xlink:to="hbio_CaseInSuffolkSuperiorCourtMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_LitigationCaseAxis" xlink:to="srt_LitigationCaseTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_ConvertiblePreferredStockMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_InvestmentIssuerNameAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentIssuerNameDomain" xlink:to="hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_InvestmentIssuerNameAxis" xlink:to="us-gaap_InvestmentIssuerNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_LegalMattersAndContingenciesTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-17-product-line-disposition" xlink:href="hbio-20231231.xsd#statement-note-17-product-line-disposition" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-17-product-line-disposition" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_HoeferProductLineMember" xlink:label="hbio_HoeferProductLineMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationAxis" xlink:label="us-gaap_DisposalGroupClassificationAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:label="us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:to="hbio_HoeferProductLineMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DisposalGroupClassificationAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupClassificationDomain" xlink:to="us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-18-government-assistance" xlink:href="hbio-20231231.xsd#statement-note-18-government-assistance" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-18-government-assistance" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_PaycheckProtectionProgramCaresActMember" xlink:label="hbio_PaycheckProtectionProgramCaresActMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:label="hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTextBlock" xlink:label="us-gaap_GovernmentAssistanceTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeAxis" xlink:label="us-gaap_GovernmentAssistanceTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeDomain" xlink:label="us-gaap_GovernmentAssistanceTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeDomain" xlink:label="us-gaap_GovernmentAssistanceTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GovernmentAssistanceTypeAxis" xlink:to="us-gaap_GovernmentAssistanceTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_GovernmentAssistanceTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GovernmentAssistanceTypeDomain" xlink:to="hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GovernmentAssistanceTypeAxis" xlink:to="us-gaap_GovernmentAssistanceTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GovernmentAssistanceTypeDomain" xlink:to="hbio_PaycheckProtectionProgramCaresActMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_GovernmentAssistanceTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-significant-accounting-policies-policies" xlink:href="hbio-20231231.xsd#statement-significant-accounting-policies-policies" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-significant-accounting-policies-policies" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ComprehensiveIncomeLossPolicyTextBlock" xlink:label="hbio_ComprehensiveIncomeLossPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountingPoliciesAbstract" xlink:label="us-gaap_AccountingPoliciesAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:label="us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommitmentsAndContingenciesPolicyTextBlock" xlink:label="us-gaap_CommitmentsAndContingenciesPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConsolidationPolicyTextBlock" xlink:label="us-gaap_ConsolidationPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativesPolicyTextBlock" xlink:label="us-gaap_DerivativesPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerSharePolicyTextBlock" xlink:label="us-gaap_EarningsPerSharePolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueOfFinancialInstrumentsPolicy" xlink:label="us-gaap_FairValueOfFinancialInstrumentsPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ForeignCurrencyTransactionsAndTranslationsPolicyTextBlock" xlink:label="us-gaap_ForeignCurrencyTransactionsAndTranslationsPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GoodwillAndIntangibleAssetsGoodwillPolicy" xlink:label="us-gaap_GoodwillAndIntangibleAssetsGoodwillPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:label="us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxPolicyTextBlock" xlink:label="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IntangibleAssetsFiniteLivedPolicy" xlink:label="us-gaap_IntangibleAssetsFiniteLivedPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InternalUseSoftwarePolicy" xlink:label="us-gaap_InternalUseSoftwarePolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryPolicyTextBlock" xlink:label="us-gaap_InventoryPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeLeasesPolicyTextBlock" xlink:label="us-gaap_LesseeLeasesPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MarketableSecuritiesPolicy" xlink:label="us-gaap_MarketableSecuritiesPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:label="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PolicyTextBlockAbstract" xlink:label="us-gaap_PolicyTextBlockAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PriorPeriodReclassificationAdjustmentDescription" xlink:label="us-gaap_PriorPeriodReclassificationAdjustmentDescription" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:label="us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ReceivablesTradeAndOtherAccountsReceivableAllowanceForDoubtfulAccountsPolicy" xlink:label="us-gaap_ReceivablesTradeAndOtherAccountsReceivableAllowanceForDoubtfulAccountsPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerPolicyTextBlock" xlink:label="us-gaap_RevenueFromContractWithCustomerPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:label="us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationOptionAndIncentivePlansPolicy" xlink:label="us-gaap_ShareBasedCompensationOptionAndIncentivePlansPolicy" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityPolicyTextBlock" xlink:label="us-gaap_StockholdersEquityPolicyTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UseOfEstimates" xlink:label="us-gaap_UseOfEstimates" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PolicyTextBlockAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PolicyTextBlockAbstract" xlink:to="us-gaap_AccountingPoliciesAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ConsolidationPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_UseOfEstimates" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_MarketableSecuritiesPolicy" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ReceivablesTradeAndOtherAccountsReceivableAllowanceForDoubtfulAccountsPolicy" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_InventoryPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_LesseeLeasesPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ForeignCurrencyTransactionsAndTranslationsPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_EarningsPerSharePolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="hbio_ComprehensiveIncomeLossPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_RevenueFromContractWithCustomerPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_InternalUseSoftwarePolicy" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_IntangibleAssetsFiniteLivedPolicy" xlink:type="arc"/>
<link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_GoodwillAndIntangibleAssetsGoodwillPolicy" xlink:type="arc"/>
<link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_DerivativesPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_FairValueOfFinancialInstrumentsPolicy" xlink:type="arc"/>
<link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ShareBasedCompensationOptionAndIncentivePlansPolicy" xlink:type="arc"/>
<link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_StockholdersEquityPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_CommitmentsAndContingenciesPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="arc"/>
<link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_PriorPeriodReclassificationAdjustmentDescription" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-tables" xlink:href="hbio-20231231.xsd#statement-note-2-summary-of-significant-accounting-policies-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_PropertyPlantAndEquipmentUsefulLivesTableTextBlock" xlink:label="hbio_PropertyPlantAndEquipmentUsefulLivesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfEarningsPerShareBasicAndDilutedTableTextBlock" xlink:label="us-gaap_ScheduleOfEarningsPerShareBasicAndDilutedTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="hbio_PropertyPlantAndEquipmentUsefulLivesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEarningsPerShareBasicAndDilutedTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss-tables" xlink:href="hbio-20231231.xsd#statement-note-3-accumulated-other-comprehensive-loss-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:label="us-gaap_ScheduleOfAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-tables" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfGoodwillTextBlock" xlink:label="us-gaap_ScheduleOfGoodwillTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfIntangibleAssetsAndGoodwillTableTextBlock" xlink:label="us-gaap_ScheduleOfIntangibleAssetsAndGoodwillTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleofFiniteLivedIntangibleAssetsFutureAmortizationExpenseTableTextBlock" xlink:label="us-gaap_ScheduleofFiniteLivedIntangibleAssetsFutureAmortizationExpenseTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfGoodwillTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfIntangibleAssetsAndGoodwillTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleofFiniteLivedIntangibleAssetsFutureAmortizationExpenseTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-tables" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongLivedAssetsByGeographicAreasTableTextBlock" xlink:label="us-gaap_LongLivedAssetsByGeographicAreasTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherCurrentLiabilitiesTableTextBlock" xlink:label="us-gaap_OtherCurrentLiabilitiesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:label="us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfInventoryCurrentTableTextBlock" xlink:label="us-gaap_ScheduleOfInventoryCurrentTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfInventoryCurrentTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_OtherCurrentLiabilitiesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_LongLivedAssetsByGeographicAreasTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs-tables" xlink:href="hbio-20231231.xsd#statement-note-6-restructuring-and-other-exit-costs-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfRestructuringAndRelatedCostsTextBlock" xlink:label="us-gaap_ScheduleOfRestructuringAndRelatedCostsTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfRestructuringAndRelatedCostsTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-tables" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanPlanAssetsCategoryTableTextBlock" xlink:label="us-gaap_DefinedBenefitPlanPlanAssetsCategoryTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAllocationOfPlanAssetsTableTextBlock" xlink:label="us-gaap_ScheduleOfAllocationOfPlanAssetsTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAmountsRecognizedInBalanceSheetTableTextBlock" xlink:label="us-gaap_ScheduleOfAmountsRecognizedInBalanceSheetTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAssumptionsUsedTableTextBlock" xlink:label="us-gaap_ScheduleOfAssumptionsUsedTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfChangesInFairValueOfPlanAssetsTableTextBlock" xlink:label="us-gaap_ScheduleOfChangesInFairValueOfPlanAssetsTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfChangesInProjectedBenefitObligationsTableTextBlock" xlink:label="us-gaap_ScheduleOfChangesInProjectedBenefitObligationsTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfCostsOfRetirementPlansTableTextBlock" xlink:label="us-gaap_ScheduleOfCostsOfRetirementPlansTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfCostsOfRetirementPlansTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfChangesInFairValueOfPlanAssetsTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfChangesInProjectedBenefitObligationsTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAmountsRecognizedInBalanceSheetTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAssumptionsUsedTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAllocationOfPlanAssetsTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_DefinedBenefitPlanPlanAssetsCategoryTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-tables" xlink:href="hbio-20231231.xsd#statement-note-8-leases-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SupplementalBalanceSheetInformatonRelatedToOperatingLeasesTableTextBlock" xlink:label="hbio_SupplementalBalanceSheetInformatonRelatedToOperatingLeasesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SupplementalCashFlowInformationRelatedToOperatingLeasesTableTextBlock" xlink:label="hbio_SupplementalCashFlowInformationRelatedToOperatingLeasesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseCostTableTextBlock" xlink:label="us-gaap_LeaseCostTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_LeaseCostTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="hbio_SupplementalBalanceSheetInformatonRelatedToOperatingLeasesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="hbio_SupplementalCashFlowInformationRelatedToOperatingLeasesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-tables" xlink:href="hbio-20231231.xsd#statement-note-9-longterm-debt-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfDebtTableTextBlock" xlink:label="us-gaap_ScheduleOfDebtTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfMaturitiesOfLongTermDebtTableTextBlock" xlink:label="us-gaap_ScheduleOfMaturitiesOfLongTermDebtTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDebtTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfMaturitiesOfLongTermDebtTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-tables" xlink:href="hbio-20231231.xsd#statement-note-10-derivatives-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfCashFlowHedgesIncludedInAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:label="us-gaap_ScheduleOfCashFlowHedgesIncludedInAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfDerivativeInstrumentsTextBlock" xlink:label="us-gaap_ScheduleOfDerivativeInstrumentsTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDerivativeInstrumentsTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfCashFlowHedgesIncludedInAccumulatedOtherComprehensiveIncomeLossTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements-tables" xlink:href="hbio-20231231.xsd#statement-note-11-fair-value-measurements-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock" xlink:label="us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-tables" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_BlackScholesOptionPricingModelMember" xlink:label="hbio_BlackScholesOptionPricingModelMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfStockOptionsRollForwardTableTextBlock" xlink:label="us-gaap_ScheduleOfStockOptionsRollForwardTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueAxis" xlink:label="us-gaap_ValuationTechniqueAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ValuationTechniqueAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ValuationTechniqueDomain" xlink:to="hbio_BlackScholesOptionPricingModelMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfStockOptionsRollForwardTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-tables" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AllowanceForCreditLossesOnFinancingReceivablesTableTextBlock" xlink:label="us-gaap_AllowanceForCreditLossesOnFinancingReceivablesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:label="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisaggregationOfRevenueTableTextBlock" xlink:label="us-gaap_DisaggregationOfRevenueTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromExternalCustomersByGeographicAreasTableTextBlock" xlink:label="us-gaap_RevenueFromExternalCustomersByGeographicAreasTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfProductWarrantyLiabilityTableTextBlock" xlink:label="us-gaap_ScheduleOfProductWarrantyLiabilityTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_DisaggregationOfRevenueTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_RevenueFromExternalCustomersByGeographicAreasTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_AllowanceForCreditLossesOnFinancingReceivablesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfProductWarrantyLiabilityTableTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-tables" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-tables" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-tables" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfComponentsOfIncomeTaxExpenseBenefitTableTextBlock" xlink:label="us-gaap_ScheduleOfComponentsOfIncomeTaxExpenseBenefitTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:label="us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:label="us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfIncomeBeforeIncomeTaxDomesticAndForeignTableTextBlock" xlink:label="us-gaap_ScheduleOfIncomeBeforeIncomeTaxDomesticAndForeignTableTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SummaryOfIncomeTaxContingenciesTextBlock" xlink:label="us-gaap_SummaryOfIncomeTaxContingenciesTextBlock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfComponentsOfIncomeTaxExpenseBenefitTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfIncomeBeforeIncomeTaxDomesticAndForeignTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_SummaryOfIncomeTaxContingenciesTextBlock" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-details-textual" xlink:href="hbio-20231231.xsd#statement-note-2-summary-of-significant-accounting-policies-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_CustomerPaymentsTerm" xlink:label="hbio_CustomerPaymentsTerm" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_PercentageOfCashAndCashEquivalentsHeldByForeignSubsidiaries" xlink:label="hbio_PercentageOfCashAndCashEquivalentsHeldByForeignSubsidiaries" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementAdjustmentMember" xlink:label="srt_RestatementAdjustmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementAxis" xlink:label="srt_RestatementAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:label="us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GoodwillImpairmentLoss" xlink:label="us-gaap_GoodwillImpairmentLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ImpairmentOfLongLivedAssetsHeldForUse" xlink:label="us-gaap_ImpairmentOfLongLivedAssetsHeldForUse" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="us-gaap_PreferredStockSharesIssued" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RestatementAxis" xlink:to="srt_RestatementDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RestatementAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RestatementDomain" xlink:to="srt_RestatementAdjustmentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RestatementAxis" xlink:to="srt_RestatementDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_PercentageOfCashAndCashEquivalentsHeldByForeignSubsidiaries" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_CustomerPaymentsTerm" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GoodwillImpairmentLoss" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ImpairmentOfLongLivedAssetsHeldForUse" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesIssued" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-details-textual" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_WriteoffOfFullyAmortizedIntangibleAssetsRelatedToExistingTechnologyAndDiscontinuedProductLines" xlink:label="hbio_WriteoffOfFullyAmortizedIntangibleAssetsRelatedToExistingTechnologyAndDiscontinuedProductLines" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AmortizationOfIntangibleAssets" xlink:label="us-gaap_AmortizationOfIntangibleAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CapitalizedComputerSoftwareNet" xlink:label="us-gaap_CapitalizedComputerSoftwareNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_WriteoffOfFullyAmortizedIntangibleAssetsRelatedToExistingTechnologyAndDiscontinuedProductLines" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CapitalizedComputerSoftwareNet" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AmortizationOfIntangibleAssets" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-details-textual" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_FullyDepreciatedAndDisposedOfPropertyAndEquipmentRemovedFromFixedAssetRecords" xlink:label="hbio_FullyDepreciatedAndDisposedOfPropertyAndEquipmentRemovedFromFixedAssetRecords" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Depreciation" xlink:label="us-gaap_Depreciation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_Depreciation" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_FullyDepreciatedAndDisposedOfPropertyAndEquipmentRemovedFromFixedAssetRecords" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-details-textual" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsFiveFiscalYearsThereafter" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsFiveFiscalYearsThereafter" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsNextTwelveMonths" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsNextTwelveMonths" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFive" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFive" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFour" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFour" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearThree" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearThree" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearTwo" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearTwo" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsNextFiscalYear" xlink:label="us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsNextFiscalYear" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedContributionPlanCostRecognized" xlink:label="us-gaap_DefinedContributionPlanCostRecognized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedContributionPlanCostRecognized" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsNextFiscalYear" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsNextTwelveMonths" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearTwo" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearThree" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFour" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsYearFive" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedFutureBenefitPaymentsFiveFiscalYearsThereafter" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-details-textual" xlink:href="hbio-20231231.xsd#statement-note-9-longterm-debt-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DebtInstrumentCurrentMaturitiesExcessCashFlowSweep" xlink:label="hbio_DebtInstrumentCurrentMaturitiesExcessCashFlowSweep" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DebtInstrumentInterestRateStateFloorPercentage" xlink:label="hbio_DebtInstrumentInterestRateStateFloorPercentage" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DebtInstrumentQuarterlyPaymentThereafter" xlink:label="hbio_DebtInstrumentQuarterlyPaymentThereafter" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SecuredOvernightFinancingRateSofrMember" xlink:label="hbio_SecuredOvernightFinancingRateSofrMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SwinglineLoanFacilityMember" xlink:label="hbio_SwinglineLoanFacilityMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TermLoanMember" xlink:label="hbio_TermLoanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheABRLoanMember" xlink:label="hbio_TheABRLoanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheCreditAgreementMember" xlink:label="hbio_TheCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheLendersMember" xlink:label="hbio_TheLendersMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:label="hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BaseRateMember" xlink:label="us-gaap_BaseRateMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityAxis" xlink:label="us-gaap_CreditFacilityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:label="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="us-gaap_DebtInstrumentCarryingAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredFinanceCostsNet" xlink:label="us-gaap_DeferredFinanceCostsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LetterOfCreditMember" xlink:label="us-gaap_LetterOfCreditMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityAxis" xlink:label="us-gaap_LineOfCreditFacilityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityInterestRateAtPeriodEnd" xlink:label="us-gaap_LineOfCreditFacilityInterestRateAtPeriodEnd" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:label="us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:label="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditFacilityRemainingBorrowingCapacity" xlink:label="us-gaap_LineOfCreditFacilityRemainingBorrowingCapacity" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevolvingCreditFacilityMember" xlink:label="us-gaap_RevolvingCreditFacilityMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LineOfCreditFacilityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LineOfCreditFacilityLenderDomain" xlink:to="hbio_TheLendersMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LongtermDebtTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="hbio_TermLoanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="hbio_TheCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_CreditFacilityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="us-gaap_RevolvingCreditFacilityMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="us-gaap_LetterOfCreditMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="hbio_SwinglineLoanFacilityMember" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VariableRateAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="hbio_SecuredOvernightFinancingRateSofrMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_BaseRateMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="hbio_TheSOFRLoanAndPricingGridCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="hbio_TheABRLoanMember" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentCarryingAmount" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredFinanceCostsNet" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LineOfCreditFacilityRemainingBorrowingCapacity" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DebtInstrumentInterestRateStateFloorPercentage" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LineOfCreditFacilityInterestRateAtPeriodEnd" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DebtInstrumentQuarterlyPaymentThereafter" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DebtInstrumentCurrentMaturitiesExcessCashFlowSweep" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-details-textual" xlink:href="hbio-20231231.xsd#statement-note-10-derivatives-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TheCreditAgreementMember" xlink:label="hbio_TheCreditAgreementMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeInstrumentRiskAxis" xlink:label="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeNotionalAmount" xlink:label="us-gaap_DerivativeNotionalAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestRateSwapMember" xlink:label="us-gaap_InterestRateSwapMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativeContractTypeDomain" xlink:to="us-gaap_InterestRateSwapMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="hbio_TheCreditAgreementMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DerivativeNotionalAmount" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-details-textual" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EmployeeStockOwnershipPlanSharesAvailableForIssuance" xlink:label="hbio_EmployeeStockOwnershipPlanSharesAvailableForIssuance" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EmployeeStockPurchasePlanMember" xlink:label="hbio_EmployeeStockPurchasePlanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_MaximumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:label="hbio_MaximumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_MinimumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:label="hbio_MinimumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TwentyTwentyOneIncentivePlanMember" xlink:label="hbio_TwentyTwentyOneIncentivePlanMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_VestingRateOfTheTargetedShareholderReturn" xlink:label="hbio_VestingRateOfTheTargetedShareholderReturn" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardDiscountFromMarketPriceOfferingDate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardDiscountFromMarketPriceOfferingDate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="hbio_TwentyTwentyOneIncentivePlanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="hbio_EmployeeStockPurchasePlanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EmployeeStockOwnershipPlanSharesAvailableForIssuance" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_VestingRateOfTheTargetedShareholderReturn" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_MinimumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_MaximumVestingRateOfTheTargetedShareholderReturnPercentage" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharePrice" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardDiscountFromMarketPriceOfferingDate" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-details-textual" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiabilityRevenueRecognized" xlink:label="us-gaap_ContractWithCustomerLiabilityRevenueRecognized" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ContractWithCustomerLiabilityRevenueRecognized" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-details-textual" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_Expire2024Member" xlink:label="hbio_Expire2024Member" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ExpireBetween2024And2043Member" xlink:label="hbio_ExpireBetween2024And2043Member" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ResearchAndDevelopmentTaxCreditCarryforwardsOffsetByReserveForUncertainTaxPositions" xlink:label="hbio_ResearchAndDevelopmentTaxCreditCarryforwardsOffsetByReserveForUncertainTaxPositions" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesAxis" xlink:label="srt_ConsolidatedEntitiesAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesDomain" xlink:label="srt_ConsolidatedEntitiesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ConsolidatedEntitiesDomain" xlink:label="srt_ConsolidatedEntitiesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SubsidiariesMember" xlink:label="srt_SubsidiariesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxCreditCarryforwards" xlink:label="us-gaap_DeferredTaxAssetsTaxCreditCarryforwards" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:label="us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:label="us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ForeignCountryMember" xlink:label="us-gaap_ForeignCountryMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonUsMember" xlink:label="us-gaap_NonUsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLossCarryforwards" xlink:label="us-gaap_OperatingLossCarryforwards" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchMember" xlink:label="us-gaap_ResearchMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StateAndLocalJurisdictionMember" xlink:label="us-gaap_StateAndLocalJurisdictionMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAmount" xlink:label="us-gaap_TaxCreditCarryforwardAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAxis" xlink:label="us-gaap_TaxCreditCarryforwardAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodAxis" xlink:label="us-gaap_TaxPeriodAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodDomain" xlink:label="us-gaap_TaxPeriodDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxPeriodDomain" xlink:label="us-gaap_TaxPeriodDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefitsThatWouldImpactEffectiveTaxRate" xlink:label="us-gaap_UnrecognizedTaxBenefitsThatWouldImpactEffectiveTaxRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:label="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ConsolidatedEntitiesAxis" xlink:to="srt_ConsolidatedEntitiesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxPeriodAxis" xlink:to="us-gaap_TaxPeriodDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_StateAndLocalJurisdictionMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxPeriodAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxPeriodDomain" xlink:to="hbio_ExpireBetween2024And2043Member" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxPeriodAxis" xlink:to="us-gaap_TaxPeriodDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_ForeignCountryMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxPeriodDomain" xlink:to="hbio_Expire2024Member" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxCreditCarryforwardAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxCreditCarryforwardNameDomain" xlink:to="us-gaap_ResearchMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_StatementGeographicalAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="us-gaap_NonUsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain-default" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ConsolidatedEntitiesAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ConsolidatedEntitiesDomain" xlink:to="srt_SubsidiariesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ConsolidatedEntitiesAxis" xlink:to="srt_ConsolidatedEntitiesDomain-default" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLossCarryforwards" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_ResearchAndDevelopmentTaxCreditCarryforwardsOffsetByReserveForUncertainTaxPositions" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxCreditCarryforwards" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TaxCreditCarryforwardAmount" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsThatWouldImpactEffectiveTaxRate" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-15-commitments-and-contingent-liabilities-details-textual" xlink:href="hbio-20231231.xsd#statement-note-15-commitments-and-contingent-liabilities-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-15-commitments-and-contingent-liabilities-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_IndemnificationAgreementsMember" xlink:label="hbio_IndemnificationAgreementsMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_LiabilityForContingentIndemnificationObligations" xlink:label="hbio_LiabilityForContingentIndemnificationObligations" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_DirectorMember" xlink:label="srt_DirectorMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_TitleOfIndividualAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="srt_DirectorMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="hbio_IndemnificationAgreementsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_LiabilityForContingentIndemnificationObligations" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-16-litigation-settlement-details-textual" xlink:href="hbio-20231231.xsd#statement-note-16-litigation-settlement-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-16-litigation-settlement-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_CaseInSuffolkSuperiorCourtMember" xlink:label="hbio_CaseInSuffolkSuperiorCourtMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:label="hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_PreferredStockBookValueInclusiveOfAccruedDividends" xlink:label="hbio_PreferredStockBookValueInclusiveOfAccruedDividends" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_StockIssuedDuringPeriodSharesIssuedForSettlement" xlink:label="hbio_StockIssuedDuringPeriodSharesIssuedForSettlement" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_StockIssuedDuringPeriodValueIssuedForSettlement" xlink:label="hbio_StockIssuedDuringPeriodValueIssuedForSettlement" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseAxis" xlink:label="srt_LitigationCaseAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseTypeDomain" xlink:label="srt_LitigationCaseTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_LitigationCaseTypeDomain" xlink:label="srt_LitigationCaseTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConvertiblePreferredStockMember" xlink:label="us-gaap_ConvertiblePreferredStockMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquitySecuritiesFvNiUnrealizedGainLoss" xlink:label="us-gaap_EquitySecuritiesFvNiUnrealizedGainLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquitySecuritiesWithoutReadilyDeterminableFairValueAmount" xlink:label="us-gaap_EquitySecuritiesWithoutReadilyDeterminableFairValueAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameAxis" xlink:label="us-gaap_InvestmentIssuerNameAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameDomain" xlink:label="us-gaap_InvestmentIssuerNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentIssuerNameDomain" xlink:label="us-gaap_InvestmentIssuerNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InvestmentOwnedAtFairValue" xlink:label="us-gaap_InvestmentOwnedAtFairValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_LitigationCaseAxis" xlink:to="srt_LitigationCaseTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_InvestmentIssuerNameAxis" xlink:to="us-gaap_InvestmentIssuerNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_LitigationCaseAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_LitigationCaseTypeDomain" xlink:to="hbio_CaseInSuffolkSuperiorCourtMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_LitigationCaseAxis" xlink:to="srt_LitigationCaseTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_ConvertiblePreferredStockMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_InvestmentIssuerNameAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentIssuerNameDomain" xlink:to="hbio_HarvardApparatusRegenerativeTechnologyIncHartMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_InvestmentIssuerNameAxis" xlink:to="us-gaap_InvestmentIssuerNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_StockIssuedDuringPeriodSharesIssuedForSettlement" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_StockIssuedDuringPeriodValueIssuedForSettlement" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EquitySecuritiesWithoutReadilyDeterminableFairValueAmount" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_PreferredStockBookValueInclusiveOfAccruedDividends" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InvestmentOwnedAtFairValue" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EquitySecuritiesFvNiUnrealizedGainLoss" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-17-product-line-disposition-details-textual" xlink:href="hbio-20231231.xsd#statement-note-17-product-line-disposition-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-17-product-line-disposition-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_HoeferProductLineMember" xlink:label="hbio_HoeferProductLineMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetsOfDisposalGroupIncludingDiscontinuedOperation" xlink:label="us-gaap_AssetsOfDisposalGroupIncludingDiscontinuedOperation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationAxis" xlink:label="us-gaap_DisposalGroupClassificationAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:label="us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:label="us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GainLossOnSaleOfBusiness" xlink:label="us-gaap_GainLossOnSaleOfBusiness" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:to="hbio_HoeferProductLineMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DisposalGroupClassificationAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupClassificationDomain" xlink:to="us-gaap_DisposalGroupDisposedOfBySaleNotDiscontinuedOperationsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsOfDisposalGroupIncludingDiscontinuedOperation" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GainLossOnSaleOfBusiness" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-18-government-assistance-details-textual" xlink:href="hbio-20231231.xsd#statement-note-18-government-assistance-details-textual" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-18-government-assistance-details-textual" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_PaycheckProtectionProgramCaresActMember" xlink:label="hbio_PaycheckProtectionProgramCaresActMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:label="hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceAmount" xlink:label="us-gaap_GovernmentAssistanceAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeAxis" xlink:label="us-gaap_GovernmentAssistanceTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeDomain" xlink:label="us-gaap_GovernmentAssistanceTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GovernmentAssistanceTypeDomain" xlink:label="us-gaap_GovernmentAssistanceTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GovernmentAssistanceTypeAxis" xlink:to="us-gaap_GovernmentAssistanceTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_GovernmentAssistanceTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GovernmentAssistanceTypeDomain" xlink:to="hbio_ProgramsEstablishedToOffsetTheImpactOfCOVIDMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GovernmentAssistanceTypeAxis" xlink:to="us-gaap_GovernmentAssistanceTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GovernmentAssistanceTypeDomain" xlink:to="hbio_PaycheckProtectionProgramCaresActMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GovernmentAssistanceAmount" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-property-plant-and-equipment-details" xlink:href="hbio-20231231.xsd#statement-note-2-summary-of-significant-accounting-policies-property-plant-and-equipment-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-property-plant-and-equipment-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ComputerEquipmentAndSoftwareMember" xlink:label="hbio_ComputerEquipmentAndSoftwareMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MachineryAndEquipmentMember" xlink:label="us-gaap_MachineryAndEquipmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:label="us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_MachineryAndEquipmentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="hbio_ComputerEquipmentAndSoftwareMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-future-amortization-expense-of-intangible-assets-details" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets-future-amortization-expense-of-intangible-assets-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-future-amortization-expense-of-intangible-assets-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsNet" xlink:label="us-gaap_FiniteLivedIntangibleAssetsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs-restructuring-and-other-exit-charges-details" xlink:href="hbio-20231231.xsd#statement-note-6-restructuring-and-other-exit-costs-restructuring-and-other-exit-charges-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-6-restructuring-and-other-exit-costs-restructuring-and-other-exit-charges-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_CostOfRevenuesMember" xlink:label="hbio_CostOfRevenuesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeSeveranceMember" xlink:label="us-gaap_EmployeeSeveranceMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherRestructuringMember" xlink:label="us-gaap_OtherRestructuringMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsForRestructuring" xlink:label="us-gaap_PaymentsForRestructuring" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestructuringCharges" xlink:label="us-gaap_RestructuringCharges" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestructuringCostAndReserveAxis" xlink:label="us-gaap_RestructuringCostAndReserveAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestructuringReserve" xlink:label="us-gaap_RestructuringReserve" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestructuringReserveSettledWithoutCash2" xlink:label="us-gaap_RestructuringReserveSettledWithoutCash2" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfRestructuringDomain" xlink:label="us-gaap_TypeOfRestructuringDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfRestructuringDomain" xlink:label="us-gaap_TypeOfRestructuringDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RestructuringCostAndReserveAxis" xlink:to="us-gaap_TypeOfRestructuringDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RestructuringCostAndReserveAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TypeOfRestructuringDomain" xlink:to="hbio_CostOfRevenuesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RestructuringCostAndReserveAxis" xlink:to="us-gaap_TypeOfRestructuringDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TypeOfRestructuringDomain" xlink:to="us-gaap_EmployeeSeveranceMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TypeOfRestructuringDomain" xlink:to="us-gaap_OtherRestructuringMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RestructuringReserve" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RestructuringCharges" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RestructuringReserveSettledWithoutCash2" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PaymentsForRestructuring" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-fair-value-of-plan-assets-by-fair-value-hierarchy-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-fair-value-of-plan-assets-by-fair-value-hierarchy-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-fair-value-of-plan-assets-by-fair-value-hierarchy-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-9-long-term-debt-debt-maturity-details" xlink:href="hbio-20231231.xsd#statement-note-9-long-term-debt-debt-maturity-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-9-long-term-debt-debt-maturity-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="us-gaap_DebtInstrumentCarryingAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInNextTwelveMonths" xlink:label="us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInNextTwelveMonths" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInYearTwo" xlink:label="us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInYearTwo" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInNextTwelveMonths" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LongTermDebtMaturitiesRepaymentsOfPrincipalInYearTwo" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentCarryingAmount" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-black-scholes-assumptions-details" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-black-scholes-assumptions-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-black-scholes-assumptions-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_BlackScholesOptionPricingModelMember" xlink:label="hbio_BlackScholesOptionPricingModelMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsCorrelationCoefficient" xlink:label="hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsCorrelationCoefficient" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsLiquidityDiscount" xlink:label="hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsLiquidityDiscount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueAxis" xlink:label="us-gaap_ValuationTechniqueAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain-default" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ValuationTechniqueAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ValuationTechniqueDomain" xlink:to="hbio_BlackScholesOptionPricingModelMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsCorrelationCoefficient" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_SharebasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsLiquidityDiscount" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-stockbased-payment-awards-details" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-stockbased-payment-awards-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-stockbased-payment-awards-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-unrecognized-tax-benefits-details" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-unrecognized-tax-benefits-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-unrecognized-tax-benefits-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_UnrecognizedTaxBenefitsOtherIncreaseDecrease" xlink:label="hbio_UnrecognizedTaxBenefitsOtherIncreaseDecrease" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefits" xlink:label="us-gaap_UnrecognizedTaxBenefits" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefitsDecreasesResultingFromPriorPeriodTaxPositions" xlink:label="us-gaap_UnrecognizedTaxBenefitsDecreasesResultingFromPriorPeriodTaxPositions" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:label="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromPriorPeriodTaxPositions" xlink:label="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromPriorPeriodTaxPositions" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefits" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromPriorPeriodTaxPositions" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsDecreasesResultingFromPriorPeriodTaxPositions" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_UnrecognizedTaxBenefitsOtherIncreaseDecrease" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-schedule-of-basic-and-diluted-earnings-per-share-details" xlink:href="hbio-20231231.xsd#statement-note-2-summary-of-significant-accounting-policies-schedule-of-basic-and-diluted-earnings-per-share-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-2-summary-of-significant-accounting-policies-schedule-of-basic-and-diluted-earnings-per-share-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareDiluted" xlink:label="us-gaap_EarningsPerShareDiluted" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncrementalCommonSharesAttributableToShareBasedPaymentArrangements" xlink:label="us-gaap_IncrementalCommonSharesAttributableToShareBasedPaymentArrangements" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding" xlink:label="us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncrementalCommonSharesAttributableToShareBasedPaymentArrangements" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareDiluted" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-change-in-carrying-amount-of-goodwill-details" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets-change-in-carrying-amount-of-goodwill-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-change-in-carrying-amount-of-goodwill-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Goodwill" xlink:label="us-gaap_Goodwill" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GoodwillForeignCurrencyTranslationGainLoss" xlink:label="us-gaap_GoodwillForeignCurrencyTranslationGainLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_Goodwill" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GoodwillForeignCurrencyTranslationGainLoss" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-inventories-details" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-inventories-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-inventories-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryFinishedGoodsNetOfReserves" xlink:label="us-gaap_InventoryFinishedGoodsNetOfReserves" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryNet" xlink:label="us-gaap_InventoryNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryRawMaterialsNetOfReserves" xlink:label="us-gaap_InventoryRawMaterialsNetOfReserves" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryWorkInProcessNetOfReserves" xlink:label="us-gaap_InventoryWorkInProcessNetOfReserves" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InventoryFinishedGoodsNetOfReserves" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InventoryWorkInProcessNetOfReserves" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InventoryRawMaterialsNetOfReserves" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InventoryNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-property-plant-and-equipment-details" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-property-plant-and-equipment-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-property-plant-and-equipment-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ComputerEquipmentAndSoftwareMember" xlink:label="hbio_ComputerEquipmentAndSoftwareMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:label="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AutomobilesMember" xlink:label="us-gaap_AutomobilesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseholdImprovementsMember" xlink:label="us-gaap_LeaseholdImprovementsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MachineryAndEquipmentMember" xlink:label="us-gaap_MachineryAndEquipmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="us-gaap_PropertyPlantAndEquipmentGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_MachineryAndEquipmentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="hbio_ComputerEquipmentAndSoftwareMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_LeaseholdImprovementsMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_AutomobilesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-other-current-liabilities-details" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-other-current-liabilities-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-other-current-liabilities-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ContractWithCustomerLiabilityCustomerAdvancesCurrent" xlink:label="hbio_ContractWithCustomerLiabilityCustomerAdvancesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedProfessionalFeesCurrent" xlink:label="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:label="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherAccruedLiabilitiesCurrent" xlink:label="us-gaap_OtherAccruedLiabilitiesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherLiabilitiesCurrent" xlink:label="us-gaap_OtherLiabilitiesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProductWarrantyAccrualClassifiedCurrent" xlink:label="us-gaap_ProductWarrantyAccrualClassifiedCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_ContractWithCustomerLiabilityCustomerAdvancesCurrent" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProductWarrantyAccrualClassifiedCurrent" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherAccruedLiabilitiesCurrent" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherLiabilitiesCurrent" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-longlived-assets-by-geographic-area-details" xlink:href="hbio-20231231.xsd#statement-note-5-balance-sheet-information-longlived-assets-by-geographic-area-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-5-balance-sheet-information-longlived-assets-by-geographic-area-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/country/2023/country-2023.xsd#country_DE" xlink:label="country_DE" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/country/2023/country-2023.xsd#country_US" xlink:label="country_US" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_RestOfTheWorldMember" xlink:label="hbio_RestOfTheWorldMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NoncurrentAssets" xlink:label="us-gaap_NoncurrentAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_StatementGeographicalAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_US" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_DE" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="hbio_RestOfTheWorldMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NoncurrentAssets" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-defined-benefit-pension-expense-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-defined-benefit-pension-expense-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-defined-benefit-pension-expense-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses" xlink:label="us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanInterestCost" xlink:label="us-gaap_DefinedBenefitPlanInterestCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost" xlink:label="us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanInterestCost" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-recognized-in-consolidated-balance-sheets-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-recognized-in-consolidated-balance-sheets-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-recognized-in-consolidated-balance-sheets-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationAxis" xlink:label="us-gaap_BalanceSheetLocationAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:label="us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherNoncurrentLiabilitiesMember" xlink:label="us-gaap_OtherNoncurrentLiabilitiesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_BalanceSheetLocationAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_BalanceSheetLocationDomain" xlink:to="us-gaap_OtherNoncurrentLiabilitiesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-weighted-average-assumptions-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-weighted-average-assumptions-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-weighted-average-assumptions-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostDiscountRate" xlink:label="us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostDiscountRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostExpectedLongTermReturnOnAssets" xlink:label="us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostExpectedLongTermReturnOnAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostDiscountRate" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanAssumptionsUsedCalculatingNetPeriodicBenefitCostExpectedLongTermReturnOnAssets" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-fair-value-and-asset-allocations-of-pension-benefits-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-fair-value-and-asset-allocations-of-pension-benefits-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-fair-value-and-asset-allocations-of-pension-benefits-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_OtherPlanAssetsMember" xlink:label="hbio_OtherPlanAssetsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanByPlanAssetCategoriesAxis" xlink:label="us-gaap_DefinedBenefitPlanByPlanAssetCategoriesAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanCashAndCashEquivalentsMember" xlink:label="us-gaap_DefinedBenefitPlanCashAndCashEquivalentsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanEquitySecuritiesMember" xlink:label="us-gaap_DefinedBenefitPlanEquitySecuritiesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanWeightedAverageAssetAllocations" xlink:label="us-gaap_DefinedBenefitPlanWeightedAverageAssetAllocations" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DomesticCorporateDebtSecuritiesMember" xlink:label="us-gaap_DomesticCorporateDebtSecuritiesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanAssetCategoriesDomain" xlink:label="us-gaap_PlanAssetCategoriesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanAssetCategoriesDomain" xlink:label="us-gaap_PlanAssetCategoriesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DefinedBenefitPlanByPlanAssetCategoriesAxis" xlink:to="us-gaap_PlanAssetCategoriesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DefinedBenefitPlanByPlanAssetCategoriesAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanAssetCategoriesDomain" xlink:to="us-gaap_DomesticCorporateDebtSecuritiesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DefinedBenefitPlanByPlanAssetCategoriesAxis" xlink:to="us-gaap_PlanAssetCategoriesDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanAssetCategoriesDomain" xlink:to="us-gaap_DefinedBenefitPlanEquitySecuritiesMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanAssetCategoriesDomain" xlink:to="us-gaap_DefinedBenefitPlanCashAndCashEquivalentsMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanAssetCategoriesDomain" xlink:to="hbio_OtherPlanAssetsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanWeightedAverageAssetAllocations" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-lease-expense-details" xlink:href="hbio-20231231.xsd#statement-note-8-leases-lease-expense-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-lease-expense-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseCost" xlink:label="us-gaap_LeaseCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseCost" xlink:label="us-gaap_OperatingLeaseCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShortTermLeaseCost" xlink:label="us-gaap_ShortTermLeaseCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubleaseIncome" xlink:label="us-gaap_SubleaseIncome" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseCost" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShortTermLeaseCost" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SubleaseIncome" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LeaseCost" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-supplemental-balance-sheet-information-related-to-operating-leases-details" xlink:href="hbio-20231231.xsd#statement-note-8-leases-supplemental-balance-sheet-information-related-to-operating-leases-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-supplemental-balance-sheet-information-related-to-operating-leases-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:label="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:label="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiability" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-supplemental-cash-flow-information-related-to-operating-leases-details" xlink:href="hbio-20231231.xsd#statement-note-8-leases-supplemental-cash-flow-information-related-to-operating-leases-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-supplemental-cash-flow-information-related-to-operating-leases-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeasePayments" xlink:label="us-gaap_OperatingLeasePayments" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" xlink:label="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeasePayments" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-future-minimum-lease-payments-details" xlink:href="hbio-20231231.xsd#statement-note-8-leases-future-minimum-lease-payments-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-8-leases-future-minimum-lease-payments-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiability" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-breakdown-of-borrowings-details" xlink:href="hbio-20231231.xsd#statement-note-9-longterm-debt-breakdown-of-borrowings-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-9-longterm-debt-breakdown-of-borrowings-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TermLoanMember" xlink:label="hbio_TermLoanMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="us-gaap_DebtInstrumentCarryingAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredFinanceCostsCurrentNet" xlink:label="us-gaap_DeferredFinanceCostsCurrentNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredFinanceCostsNet" xlink:label="us-gaap_DeferredFinanceCostsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LineOfCreditMember" xlink:label="us-gaap_LineOfCreditMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebt" xlink:label="us-gaap_LongTermDebt" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtCurrent" xlink:label="us-gaap_LongTermDebtCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtNoncurrent" xlink:label="us-gaap_LongTermDebtNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LongtermDebtTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="hbio_TermLoanMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="us-gaap_LineOfCreditMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentCarryingAmount" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredFinanceCostsNet" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LongTermDebt" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LongTermDebtCurrent" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredFinanceCostsCurrentNet" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LongTermDebtNoncurrent" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-the-effect-of-derivatives-designated-as-cash-flow-hedging-instruments-details" xlink:href="hbio-20231231.xsd#statement-note-10-derivatives-the-effect-of-derivatives-designated-as-cash-flow-hedging-instruments-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-the-effect-of-derivatives-designated-as-cash-flow-hedging-instruments-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossReclassificationAfterTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossReclassificationAfterTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossReclassificationAfterTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements-assets-and-liabilities-measured-on-a-recurring-basis-details" xlink:href="hbio-20231231.xsd#statement-note-11-fair-value-measurements-assets-and-liabilities-measured-on-a-recurring-basis-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-11-fair-value-measurements-assets-and-liabilities-measured-on-a-recurring-basis-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeInstrumentRiskAxis" xlink:label="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeLiabilities" xlink:label="us-gaap_DerivativeLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquitySecuritiesFvNiCurrentAndNoncurrent" xlink:label="us-gaap_EquitySecuritiesFvNiCurrentAndNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestRateSwapMember" xlink:label="us-gaap_InterestRateSwapMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativeContractTypeDomain" xlink:to="us-gaap_InterestRateSwapMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EquitySecuritiesFvNiCurrentAndNoncurrent" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DerivativeLiabilities" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-stockbased-compensation-expense-details" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-stockbased-compensation-expense-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-stockbased-compensation-expense-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CostOfSalesMember" xlink:label="us-gaap_CostOfSalesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SegmentContinuingOperationsMember" xlink:label="us-gaap_SegmentContinuingOperationsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SegmentOperatingActivitiesDomain" xlink:label="us-gaap_SegmentOperatingActivitiesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SegmentOperatingActivitiesDomain" xlink:label="us-gaap_SegmentOperatingActivitiesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SellingAndMarketingExpenseMember" xlink:label="us-gaap_SellingAndMarketingExpenseMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementOperatingActivitiesSegmentAxis" xlink:label="us-gaap_StatementOperatingActivitiesSegmentAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementOperatingActivitiesSegmentAxis" xlink:to="us-gaap_SegmentOperatingActivitiesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementOperatingActivitiesSegmentAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentOperatingActivitiesDomain" xlink:to="us-gaap_SegmentContinuingOperationsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementOperatingActivitiesSegmentAxis" xlink:to="us-gaap_SegmentOperatingActivitiesDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementLocationAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_CostOfSalesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_SellingAndMarketingExpenseMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-disaggregation-of-revenue-details" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-disaggregation-of-revenue-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-disaggregation-of-revenue-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_InstrumentsEquipmentSoftwareAndAccessoriesMember" xlink:label="hbio_InstrumentsEquipmentSoftwareAndAccessoriesMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ServiceMaintenanceAndWarrantyContractsMember" xlink:label="hbio_ServiceMaintenanceAndWarrantyContractsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TimingOfTransferOfGoodOrServiceAxis" xlink:label="us-gaap_TimingOfTransferOfGoodOrServiceAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:label="us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:label="us-gaap_TimingOfTransferOfGoodOrServiceDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TransferredAtPointInTimeMember" xlink:label="us-gaap_TransferredAtPointInTimeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TransferredOverTimeMember" xlink:label="us-gaap_TransferredOverTimeMember" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TimingOfTransferOfGoodOrServiceAxis" xlink:to="us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="hbio_InstrumentsEquipmentSoftwareAndAccessoriesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TimingOfTransferOfGoodOrServiceAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:to="us-gaap_TransferredAtPointInTimeMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TimingOfTransferOfGoodOrServiceAxis" xlink:to="us-gaap_TimingOfTransferOfGoodOrServiceDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="hbio_ServiceMaintenanceAndWarrantyContractsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TimingOfTransferOfGoodOrServiceDomain" xlink:to="us-gaap_TransferredOverTimeMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-revenue-by-geographic-destination-details" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-revenue-by-geographic-destination-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-revenue-by-geographic-destination-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/country/2023/country-2023.xsd#country_US" xlink:label="country_US" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_GreaterChinaMember" xlink:label="hbio_GreaterChinaMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_RestOfTheWorldMember" xlink:label="hbio_RestOfTheWorldMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_EuropeMember" xlink:label="srt_EuropeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_StatementGeographicalAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_US" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="srt_EuropeMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="hbio_GreaterChinaMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="hbio_RestOfTheWorldMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-deferred-revenue-details" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-deferred-revenue-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-deferred-revenue-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_CustomerAdvancesMember" xlink:label="hbio_CustomerAdvancesMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:label="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ServiceMember" xlink:label="us-gaap_ServiceMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_ServiceMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="hbio_CustomerAdvancesMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-allowance-for-doubtful-accounts-details" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-allowance-for-doubtful-accounts-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-allowance-for-doubtful-accounts-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_AccountsReceivableAllowanceForCreditLossChargeoffsAndOtherReoveries" xlink:label="hbio_AccountsReceivableAllowanceForCreditLossChargeoffsAndOtherReoveries" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AllowanceForDoubtfulAccountsReceivable" xlink:label="us-gaap_AllowanceForDoubtfulAccountsReceivable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProvisionForDoubtfulAccounts" xlink:label="us-gaap_ProvisionForDoubtfulAccounts" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllowanceForDoubtfulAccountsReceivable" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProvisionForDoubtfulAccounts" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_AccountsReceivableAllowanceForCreditLossChargeoffsAndOtherReoveries" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-warranties-details" xlink:href="hbio-20231231.xsd#statement-note-13-revenues-warranties-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-13-revenues-warranties-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_StandardProductWarrantyAccrualClaims" xlink:label="hbio_StandardProductWarrantyAccrualClaims" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StandardProductWarrantyAccrual" xlink:label="us-gaap_StandardProductWarrantyAccrual" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StandardProductWarrantyAccrualWarrantiesIssued" xlink:label="us-gaap_StandardProductWarrantyAccrualWarrantiesIssued" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StandardProductWarrantyAccrual" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StandardProductWarrantyAccrualWarrantiesIssued" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_StandardProductWarrantyAccrualClaims" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-income-tax-rate-reconciliation-details" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-income-tax-rate-reconciliation-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-income-tax-rate-reconciliation-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EffectiveIncomeTaxRateReconciliationChangeInReserveForUncertainTaxPositionAmount" xlink:label="hbio_EffectiveIncomeTaxRateReconciliationChangeInReserveForUncertainTaxPositionAmount" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EffectiveIncomeTaxRateReconciliationNetOperatingLossTrueUpsAndExpirationsAmount" xlink:label="hbio_EffectiveIncomeTaxRateReconciliationNetOperatingLossTrueUpsAndExpirationsAmount" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseExecutiveCompensation" xlink:label="hbio_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseExecutiveCompensation" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EffectiveIncomeTaxRateReconciliationPermanentDifferencesNetAmount" xlink:label="hbio_EffectiveIncomeTaxRateReconciliationPermanentDifferencesNetAmount" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_EffectiveIncomeTaxRateReconciliationTaxCreditAmountDeduction" xlink:label="hbio_EffectiveIncomeTaxRateReconciliationTaxCreditAmountDeduction" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationGiltiAmount" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationGiltiAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationShareBasedCompensationExcessTaxBenefitAmount" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationShareBasedCompensationExcessTaxBenefitAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationOtherAdjustments" xlink:label="us-gaap_IncomeTaxReconciliationOtherAdjustments" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationPriorYearIncomeTaxes" xlink:label="us-gaap_IncomeTaxReconciliationPriorYearIncomeTaxes" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationStateAndLocalIncomeTaxes" xlink:label="us-gaap_IncomeTaxReconciliationStateAndLocalIncomeTaxes" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EffectiveIncomeTaxRateReconciliationPermanentDifferencesNetAmount" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseExecutiveCompensation" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationGiltiAmount" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationStateAndLocalIncomeTaxes" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationShareBasedCompensationExcessTaxBenefitAmount" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EffectiveIncomeTaxRateReconciliationTaxCreditAmountDeduction" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EffectiveIncomeTaxRateReconciliationNetOperatingLossTrueUpsAndExpirationsAmount" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_EffectiveIncomeTaxRateReconciliationChangeInReserveForUncertainTaxPositionAmount" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationPriorYearIncomeTaxes" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationOtherAdjustments" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxExpenseBenefit" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-pretax-income-from-continuing-operations-details" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-pretax-income-from-continuing-operations-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-pretax-income-from-continuing-operations-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesForeign" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesForeign" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesForeign" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-derivative-instruments-details" xlink:href="hbio-20231231.xsd#statement-note-10-derivatives-derivative-instruments-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-10-derivatives-derivative-instruments-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="us-gaap_DerivativeContractTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeFairValueOfDerivativeNet" xlink:label="us-gaap_DerivativeFairValueOfDerivativeNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeInstrumentRiskAxis" xlink:label="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DerivativeNotionalAmount" xlink:label="us-gaap_DerivativeNotionalAmount" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestRateSwapMember" xlink:label="us-gaap_InterestRateSwapMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DerivativeInstrumentRiskAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativeContractTypeDomain" xlink:to="us-gaap_InterestRateSwapMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DerivativeInstrumentRiskAxis" xlink:to="us-gaap_DerivativeContractTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DerivativeNotionalAmount" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DerivativeFairValueOfDerivativeNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-balance-sheets" xlink:href="hbio-20231231.xsd#statement-consolidated-balance-sheets" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-balance-sheets" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsPayableCurrent" xlink:label="us-gaap_AccountsPayableCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsReceivableNetCurrent" xlink:label="us-gaap_AccountsReceivableNetCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdditionalPaidInCapitalCommonStock" xlink:label="us-gaap_AdditionalPaidInCapitalCommonStock" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetsCurrent" xlink:label="us-gaap_AssetsCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="us-gaap_AssetsCurrentAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="us-gaap_CommitmentsAndContingencies" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockValue" xlink:label="us-gaap_CommonStockValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiabilityCurrent" xlink:label="us-gaap_ContractWithCustomerLiabilityCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:label="us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Goodwill" xlink:label="us-gaap_Goodwill" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:label="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InventoryNet" xlink:label="us-gaap_InventoryNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Liabilities" xlink:label="us-gaap_Liabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesCurrent" xlink:label="us-gaap_LiabilitiesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LongTermDebtNoncurrent" xlink:label="us-gaap_LongTermDebtNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherAssetsCurrent" xlink:label="us-gaap_OtherAssetsCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherAssetsNoncurrent" xlink:label="us-gaap_OtherAssetsNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherLiabilitiesCurrent" xlink:label="us-gaap_OtherLiabilitiesCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherLiabilitiesNoncurrent" xlink:label="us-gaap_OtherLiabilitiesNoncurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockValue" xlink:label="us-gaap_PreferredStockValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SecuredDebtCurrent" xlink:label="us-gaap_SecuredDebtCurrent" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityAbstract" xlink:label="us-gaap_StockholdersEquityAbstract" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsCurrentAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AccountsReceivableNetCurrent" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_InventoryNet" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_OtherAssetsCurrent" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_Goodwill" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_OtherAssetsNoncurrent" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_Assets" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesCurrentAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_SecuredDebtCurrent" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_ContractWithCustomerLiabilityCurrent" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OtherLiabilitiesCurrent" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesCurrent" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LongTermDebtNoncurrent" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OtherLiabilitiesNoncurrent" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_Liabilities" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_PreferredStockValue" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_CommonStockValue" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AdditionalPaidInCapitalCommonStock" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss-changes-in-each-component-of-other-comprehensive-loss-net-of-tax-details" xlink:href="hbio-20231231.xsd#statement-note-3-accumulated-other-comprehensive-loss-changes-in-each-component-of-other-comprehensive-loss-net-of-tax-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-3-accumulated-other-comprehensive-loss-changes-in-each-component-of-other-comprehensive-loss-net-of-tax-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember" xlink:label="us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember" xlink:label="us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedTranslationAdjustmentMember" xlink:label="us-gaap_AccumulatedTranslationAdjustmentMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedTranslationAdjustmentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-intangible-assets-details" xlink:href="hbio-20231231.xsd#statement-note-4-goodwill-and-intangible-assets-intangible-assets-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-4-goodwill-and-intangible-assets-intangible-assets-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DistributionAgreementscustomerRelationshipsMember" xlink:label="hbio_DistributionAgreementscustomerRelationshipsMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_ExistingTechnologyAndSoftwareDevelopmentMember" xlink:label="hbio_ExistingTechnologyAndSoftwareDevelopmentMember" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_TradeNamesAndPatentsMember" xlink:label="hbio_TradeNamesAndPatentsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:label="us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsGross" xlink:label="us-gaap_FiniteLivedIntangibleAssetsGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FiniteLivedIntangibleAssetsNet" xlink:label="us-gaap_FiniteLivedIntangibleAssetsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IndefiniteLivedIntangibleAssetsExcludingGoodwill" xlink:label="us-gaap_IndefiniteLivedIntangibleAssetsExcludingGoodwill" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:label="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="hbio_DistributionAgreementscustomerRelationshipsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="hbio_ExistingTechnologyAndSoftwareDevelopmentMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="hbio_TradeNamesAndPatentsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsGross" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsNet" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IndefiniteLivedIntangibleAssetsExcludingGoodwill" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-change-in-fair-value-of-plan-assets-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-change-in-fair-value-of-plan-assets-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-change-in-fair-value-of-plan-assets-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanActualReturnOnPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanActualReturnOnPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanContributionsByEmployer" xlink:label="us-gaap_DefinedBenefitPlanContributionsByEmployer" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:label="us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanPlanAssetsForeignCurrencyTranslationGainLoss" xlink:label="us-gaap_DefinedBenefitPlanPlanAssetsForeignCurrencyTranslationGainLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanActualReturnOnPlanAssets" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanContributionsByEmployer" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanPlanAssetsForeignCurrencyTranslationGainLoss" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-change-in-benefit-obligations-details" xlink:href="hbio-20231231.xsd#statement-note-7-employee-benefit-plans-change-in-benefit-obligations-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-7-employee-benefit-plans-change-in-benefit-obligations-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanActuarialGainLoss" xlink:label="us-gaap_DefinedBenefitPlanActuarialGainLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:label="us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanBenefitObligation" xlink:label="us-gaap_DefinedBenefitPlanBenefitObligation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:label="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanForeignCurrencyExchangeRateChangesBenefitObligation" xlink:label="us-gaap_DefinedBenefitPlanForeignCurrencyExchangeRateChangesBenefitObligation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanInterestCost" xlink:label="us-gaap_DefinedBenefitPlanInterestCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:label="us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PensionPlansDefinedBenefitMember" xlink:label="us-gaap_PensionPlansDefinedBenefitMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetirementPlanTypeAxis" xlink:label="us-gaap_RetirementPlanTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetirementPlanTypeDomain" xlink:label="us-gaap_RetirementPlanTypeDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetirementPlanTypeDomain" xlink:label="us-gaap_RetirementPlanTypeDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RetirementPlanTypeAxis" xlink:to="us-gaap_RetirementPlanTypeDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RetirementPlanTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RetirementPlanTypeDomain" xlink:to="us-gaap_PensionPlansDefinedBenefitMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RetirementPlanTypeAxis" xlink:to="us-gaap_RetirementPlanTypeDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanBenefitObligation" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanInterestCost" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanActuarialGainLoss" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanPlanAssetsBenefitsPaid" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanForeignCurrencyExchangeRateChangesBenefitObligation" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanFairValueOfPlanAssets" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DefinedBenefitPlanAmountsRecognizedInBalanceSheet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-activity-of-restricted-stock-and-restricted-stock-units-details" xlink:href="hbio-20231231.xsd#statement-note-12-stockbased-compensation-activity-of-restricted-stock-and-restricted-stock-units-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-12-stockbased-compensation-activity-of-restricted-stock-and-restricted-stock-units-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_MarketConditionRestrictedStockUnitsMember" xlink:label="hbio_MarketConditionRestrictedStockUnitsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="hbio_MarketConditionRestrictedStockUnitsMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-income-tax-expense-details" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-income-tax-expense-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-income-tax-expense-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CurrentFederalStateAndLocalTaxExpenseBenefit" xlink:label="us-gaap_CurrentFederalStateAndLocalTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CurrentForeignTaxExpenseBenefit" xlink:label="us-gaap_CurrentForeignTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CurrentIncomeTaxExpenseBenefit" xlink:label="us-gaap_CurrentIncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredFederalStateAndLocalTaxExpenseBenefit" xlink:label="us-gaap_DeferredFederalStateAndLocalTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredForeignIncomeTaxExpenseBenefit" xlink:label="us-gaap_DeferredForeignIncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxExpenseBenefit" xlink:label="us-gaap_DeferredIncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CurrentFederalStateAndLocalTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CurrentForeignTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CurrentIncomeTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredFederalStateAndLocalTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredForeignIncomeTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredIncomeTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxExpenseBenefit" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-deferred-tax-assets-and-liabilities-details" xlink:href="hbio-20231231.xsd#statement-note-14-income-tax-deferred-tax-assets-and-liabilities-details" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-note-14-income-tax-deferred-tax-assets-and-liabilities-details" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxAssetsEmployeeRetentionCredit" xlink:label="hbio_DeferredTaxAssetsEmployeeRetentionCredit" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxAssetsOperatingLeases" xlink:label="hbio_DeferredTaxAssetsOperatingLeases" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxAssetsOperatingLossAndTaxCreditsCarryforwards" xlink:label="hbio_DeferredTaxAssetsOperatingLossAndTaxCreditsCarryforwards" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxLiabilitiesDefiniteLivedIntangibleAssets" xlink:label="hbio_DeferredTaxLiabilitiesDefiniteLivedIntangibleAssets" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxLiabilitiesEmployeeBenefitPlans" xlink:label="hbio_DeferredTaxLiabilitiesEmployeeBenefitPlans" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxLiabilitiesIndefiniteLivedIntangibleAssets" xlink:label="hbio_DeferredTaxLiabilitiesIndefiniteLivedIntangibleAssets" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredTaxLiabilitiesOperatingLeases" xlink:label="hbio_DeferredTaxLiabilitiesOperatingLeases" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxAssetsNet" xlink:label="us-gaap_DeferredIncomeTaxAssetsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxLiabilities" xlink:label="us-gaap_DeferredIncomeTaxLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:label="us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetInterestCarryforward" xlink:label="us-gaap_DeferredTaxAssetInterestCarryforward" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsGross" xlink:label="us-gaap_DeferredTaxAssetsGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsInProcessResearchAndDevelopment" xlink:label="us-gaap_DeferredTaxAssetsInProcessResearchAndDevelopment" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsInventory" xlink:label="us-gaap_DeferredTaxAssetsInventory" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsNet" xlink:label="us-gaap_DeferredTaxAssetsNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:label="us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsAccruedLiabilities" xlink:label="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsAccruedLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsOther" xlink:label="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsOther" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxLiabilities" xlink:label="us-gaap_DeferredTaxLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxLiabilitiesOther" xlink:label="us-gaap_DeferredTaxLiabilitiesOther" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsInventory" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxAssetsOperatingLossAndTaxCreditsCarryforwards" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsInProcessResearchAndDevelopment" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxAssetsEmployeeRetentionCredit" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxAssetsOperatingLeases" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsAccruedLiabilities" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetInterestCarryforward" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccrualsOther" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsGross" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsNet" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxLiabilitiesIndefiniteLivedIntangibleAssets" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxLiabilitiesDefiniteLivedIntangibleAssets" xlink:type="arc"/>
<link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxLiabilitiesOperatingLeases" xlink:type="arc"/>
<link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="hbio_DeferredTaxLiabilitiesEmployeeBenefitPlans" xlink:type="arc"/>
<link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxLiabilitiesOther" xlink:type="arc"/>
<link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredIncomeTaxLiabilities" xlink:type="arc"/>
<link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxLiabilities" xlink:type="arc"/>
<link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredIncomeTaxAssetsNet" xlink:type="arc"/>
<link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredIncomeTaxLiabilitiesNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-cash-flows" xlink:href="hbio-20231231.xsd#statement-consolidated-statements-of-cash-flows" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-cash-flows" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_AmortizationOfIntangibleAssetsIncludingAmountsRelatedToDiscontinuedOperation" xlink:label="hbio_AmortizationOfIntangibleAssetsIncludingAmountsRelatedToDiscontinuedOperation" xlink:type="locator"/>
<link:loc xlink:href="hbio-20231231.xsd#hbio_DeferredIncomeTaxExpenseBenefitIncludingDiscontinuedOperation" xlink:label="hbio_DeferredIncomeTaxExpenseBenefitIncludingDiscontinuedOperation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AmortizationOfFinancingCosts" xlink:label="us-gaap_AmortizationOfFinancingCosts" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Depreciation" xlink:label="us-gaap_Depreciation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:label="us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GainLossOnSaleOfBusiness" xlink:label="us-gaap_GainLossOnSaleOfBusiness" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxesPaidNet" xlink:label="us-gaap_IncomeTaxesPaidNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:label="us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:label="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInEquitySecuritiesFvNi" xlink:label="us-gaap_IncreaseDecreaseInEquitySecuritiesFvNi" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInInventories" xlink:label="us-gaap_IncreaseDecreaseInInventories" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:label="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInOtherCurrentAssets" xlink:label="us-gaap_IncreaseDecreaseInOtherCurrentAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInOtherOperatingLiabilities" xlink:label="us-gaap_IncreaseDecreaseInOtherOperatingLiabilities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestPaidNet" xlink:label="us-gaap_InterestPaidNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:label="us-gaap_PaymentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsToAcquireIntangibleAssets" xlink:label="us-gaap_PaymentsToAcquireIntangibleAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:label="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromDivestitureOfBusinessesNetOfCashDivested" xlink:label="us-gaap_ProceedsFromDivestitureOfBusinessesNetOfCashDivested" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromLinesOfCredit" xlink:label="us-gaap_ProceedsFromLinesOfCredit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromStockOptionsExercised" xlink:label="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RepaymentsOfLinesOfCredit" xlink:label="us-gaap_RepaymentsOfLinesOfCredit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RepaymentsOfLongTermDebt" xlink:label="us-gaap_RepaymentsOfLongTermDebt" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_Depreciation" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="hbio_AmortizationOfIntangibleAssetsIncludingAmountsRelatedToDiscontinuedOperation" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_AmortizationOfFinancingCosts" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="hbio_DeferredIncomeTaxExpenseBenefitIncludingDiscontinuedOperation" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_IncreaseDecreaseInEquitySecuritiesFvNi" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_GainLossOnSaleOfBusiness" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInInventories" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInOtherCurrentAssets" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInOtherOperatingLiabilities" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquireIntangibleAssets" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromDivestitureOfBusinessesNetOfCashDivested" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromLinesOfCredit" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_RepaymentsOfLinesOfCredit" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_RepaymentsOfLongTermDebt" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_PaymentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_InterestPaidNet" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_IncomeTaxesPaidNet" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-operations" xlink:href="hbio-20231231.xsd#statement-consolidated-statements-of-operations" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-operations" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AmortizationOfIntangibleAssets" xlink:label="us-gaap_AmortizationOfIntangibleAssets" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CostOfGoodsAndServicesSold" xlink:label="us-gaap_CostOfGoodsAndServicesSold" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareAbstract" xlink:label="us-gaap_EarningsPerShareAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:label="us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GrossProfit" xlink:label="us-gaap_GrossProfit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestIncomeExpenseNonoperatingNet" xlink:label="us-gaap_InterestIncomeExpenseNonoperatingNet" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LitigationSettlementExpense" xlink:label="us-gaap_LitigationSettlementExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonoperatingIncomeExpense" xlink:label="us-gaap_NonoperatingIncomeExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonoperatingIncomeExpenseAbstract" xlink:label="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherNonoperatingIncomeExpense" xlink:label="us-gaap_OtherNonoperatingIncomeExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SellingAndMarketingExpense" xlink:label="us-gaap_SellingAndMarketingExpense" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingAbstract" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CostOfGoodsAndServicesSold" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GrossProfit" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SellingAndMarketingExpense" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AmortizationOfIntangibleAssets" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LitigationSettlementExpense" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingExpenses" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_InterestIncomeExpenseNonoperatingNet" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_EquitySecuritiesFvNiUnrealizedLoss" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_OtherNonoperatingIncomeExpense" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NonoperatingIncomeExpense" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeTaxExpenseBenefit" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_WeightedAverageNumberOfSharesOutstandingAbstract" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-comprehensive-loss" xlink:href="hbio-20231231.xsd#statement-consolidated-statements-of-comprehensive-loss" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-comprehensive-loss" xlink:type="extended">
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ComprehensiveIncomeNetOfTax" xlink:label="us-gaap_ComprehensiveIncomeNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationAndTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationAndTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:label="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:to="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossArisingDuringPeriodNetOfTax" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:to="us-gaap_OtherComprehensiveIncomeLossPensionAndOtherPostretirementBenefitPlansAdjustmentNetOfTax" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:to="us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossAfterReclassificationAndTax" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:to="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodIncreaseDecreaseAbstract" xlink:to="us-gaap_ComprehensiveIncomeNetOfTax" xlink:type="arc"/>
</link:definitionLink>
<link:roleRef roleURI="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-stockholders-equity" xlink:href="hbio-20231231.xsd#statement-consolidated-statements-of-stockholders-equity" xlink:type="simple"/>
<link:definitionLink xlink:role="http://www.harvardbioscience.com/20231231/role/statement-consolidated-statements-of-stockholders-equity" xlink:type="extended">
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:label="us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain-default" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharesOutstanding" xlink:label="us-gaap_SharesOutstanding" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:label="us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
<link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityOther" xlink:label="us-gaap_StockholdersEquityOther" xlink:type="locator"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain-default" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" xlink:type="arc"/>
<link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
<link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesOutstanding" xlink:type="arc"/>
<link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
<link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
<link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:type="arc"/>
<link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="arc"/>
<link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="arc"/>
<link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:type="arc"/>
<link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:type="arc"/>
<link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:type="arc"/>
<link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation" xlink:type="arc"/>
<link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="arc"/>
<link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
<link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeLossNetOfTax" xlink:type="arc"/>
<link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityOther" xlink:type="arc"/>
</link:definitionLink>
</link:linkbase>


20 Previous Filings that this Filing References

  As Of               Filer                 Filing    For·On·As Docs:Size             Issuer                      Filing Agent

 6/20/23  Harvard Bioscience Inc.           8-K:5,8,9   6/19/23   12:349K                                   Globenewswire Inc./FA
 3/09/23  Harvard Bioscience Inc.           10-K       12/31/22  112:10M                                    Globenewswire Inc./FA
 1/19/23  Harvard Bioscience Inc.           8-K:5,9     1/18/23   11:216K                                   Globenewswire Inc./FA
11/09/22  Harvard Bioscience Inc.           10-Q        9/30/22   72:7M                                     Globenewswire Inc./FA
 4/28/22  Harvard Bioscience Inc.           8-K:1,8,9   4/27/22   11:245K                                   E-Data Systems, Inc./FA
 4/07/22  Harvard Bioscience Inc.           DEF 14A     5/17/22    1:581K                                   Globenewswire Inc./FA
 3/11/22  Harvard Bioscience Inc.           10-K       12/31/21  109:9.3M                                   Globenewswire Inc./FA
 1/28/22  Harvard Bioscience Inc.           8-K:1,5,9   1/24/22   11:219K                                   E-Data Systems, Inc./FA
 5/19/21  Harvard Bioscience Inc.           8-K:5,9     5/18/21   11:424K                                   Globenewswire Inc./FA
12/23/20  Harvard Bioscience Inc.           8-K:1,2,9  12/23/20   14:1.8M                                   Globenewswire Inc./FA
 8/10/20  Harvard Bioscience Inc.           10-Q        6/30/20   79:6.9M                                   Globenewswire Inc./FA
 5/08/20  Harvard Bioscience Inc.           10-Q        3/31/20   86:4.5M                                   Globenewswire Inc./FA
 3/16/20  Harvard Bioscience Inc.           10-K       12/31/19  133:9.3M                                   Globenewswire Inc./FA
 3/06/20  Harvard Bioscience Inc.           8-K:1,9     3/02/20    2:4.8M                                   Globenewswire Inc./FA
 7/08/19  Harvard Bioscience Inc.           8-K:5,9     7/02/19    4:195K                                   Globenewswire Inc./FA
11/06/13  Harvard Bioscience Inc.           8-K:1,5,8,910/31/13    9:1M                                     Toppan Merrill/FA
 3/16/11  Harvard Bioscience Inc.           10-K       12/31/10    8:1.5M                                   Donnelley … Solutions/FA
11/01/07  Harvard Bioscience Inc.           8-K:2,5,9  10/30/07    3:199K                                   Donnelley … Solutions/FA
 3/16/06  Harvard Bioscience Inc.           10-K       12/31/05   11:2.4M                                   Toppan Merrill/FA
11/09/00  Harvard Bioscience Inc.           S-1/A                 19:1.2M                                   Toppan Merrill-FA2/FA
Top
Filing Submission 0001171843-24-001239   –   Alternative Formats (Word / Rich Text, HTML, Plain Text, et al.)

Copyright © 2024 Fran Finnegan & Company LLC – All Rights Reserved.
AboutPrivacyRedactionsHelp — Sat., May 11, 11:39:57.2pm ET