Introduction. Hepatitis B virus infection is one of the commonest occupational risks in healthcare workers. However; there is limited evidence regarding the prevalence of hepatitis in health professionals in Ethiopia. Objective. This study was aimed at assessing the prevalence of hepatitis B and associated factors in health professionals. Methods. Institution based cross-sectional study was conducted among health professionals at University of Gondar Hospital from January to February, 2015. Self-administered questionnaire was used to collect sociodemographic variables and blood sample was also taken to determine hepatitis B virus sero-status. Chi square test with 95% confidence interval (CI) was computed to assess the associations of different factors with hepatitis B infection. Result. A total of 332 health professionals (with a response rate of 92.2%) participated in the study. Most (98.5%) of health professionals were not vaccinated for hepatitis B. The prevalence of hepatitis B in health professionals at UOG hospital was found to be 4.52% (95% CI: 2.4, 6.5). Hepatitis B infection was more common among males (P value =0.0299). Conclusion. The prevalence of hepatitis B in health professionals in this study was comparable with other studies done in Ethiopia among health professionals. Males were more affected than females for hepatitis B infection. Hepatitis B virus vaccine, treatment for the infected, and training on infection prevention should be more available for healthcare workers.