BackgroundHepatitis B is a global public health problem affecting approximately 10% of the world population. Health care professionals (HCPs) are at an increased risk of acquiring hepatitis B infection due to occupational exposure. Having enough knowledge and proper attitudes toward the infection and its vaccination is crucial in preventing the infection. This study aimed to assess knowledge of and attitudes toward hepatitis B virus (HBV) infection as well as its vaccination among HCPs working in University of Gondar Hospital.MethodsAn institution-based cross-sectional study design was employed from April 1 to May 1, 2016 on 297 HCPs working at University of Gondar Hospital. A self-administered questionnaire prepared in the English language was used to collect the data. The questionnaire contained sociodemographic characteristics, knowledge, and attitude-related questions. Data were entered and analyzed using SPSS software version 20.1. Descriptive statistics, cross-tabs, and binary logistic regression were utilized. P<0.05 was used to declare association.ResultsFrom a total of 297 HCPs participated in the study 73.1% have good knowledge of HBV transmission, progress, and its vaccination. The majority (91.3%) of the respondents believe that their job puts them at risk of HBV infection. The majority of study participants (94%) believe vaccination is necessary. Medical doctors have 8.4 times better knowledge of HBV and its vaccination than other professionals (adjusted odds ratio =8.399, CI =1.536–45.936).ConclusionThe majority of HCPs working in University of Gondar Hospital have good knowledge of HBV transmission, progress, and its vaccination. The majority of HCPs believe that their job puts them at greater risk for HBV and vaccination is necessary. Knowledge of the HCPs significantly varies across professions.