Hepatitis B is one of the most common infectious diseases globally. Approximately one million hospital staffs are exposed to HBV annually in their clinical practice. This demands specific protective measures designed for the situations like biological safety. Methodology: A descriptive survey research design has been used to conduct the present study. A structured knowledge questionnaire was used to collect the data. A sample of 80 laboratory technicians working in selected Hospitals of Bagalkot was selected using convenient sampling technique. The data collected were analyzed using descriptive and inferential statistics. Results: Majority (58%) of the respondents had average knowledge on regarding modes of transmission of hepatitis B and its prevention. A significant association found between knowledge levels of laboratory technicians and their socio-demographic variables professional education (2 = 12.15, p<0.05) work experience (2 = 15. 32, p<0.05) and attending educational programme (2 = 25.61, p<0.05). Conclusion: Findings reveal that majority of laboratory techniciations have average knowledge regarding mode of transmission of Hepatitis B and its prevention. Hence future researches should consider testing the effetiveness of educational programmes in that perspectives.