Background. Hepatitis B virus is a global problem, with 66% of all the world population living in areas where there are high levels of infection. HBV is the leading risk factor for HCC globally and accounts for at least 50% of cases of HCC. Medical and health science students, being part of the health-care system, are exposed to the infection as a risk as other health-care workers when they come in contact with patients and contaminated instruments. Objective. The main aim of this study was to assess the knowledge and practice of hepatitis B virus infection prevention and its associated factors among health science students in Woldia University. Methods. Institutional-based cross-sectional study was conducted from January 30 to May 30, 2019, among health science students of Woldia University who had previous clinical attachments. Two hundred students were selected by the systematic random sampling method. Association of dependent and independent variables was computed using a bivariable and multivariable logistic regression model. P<0.05 was considered as significantly associated. Result. The study revealed that, out of 200 participants, 96 (48%) have poor knowledge, whereas 104 (52%) showed good knowledge about HBV. Regarding the practice of participants, 79 (39.5%) of the students have good practice to prevent HBV, whereas 121 (59.5%) had poor practice towards HBV infection prevention. Conclusion. Based on the current study, greater than half of the students who participated in the study have good knowledge of hepatitis B infection prevention and most of the students have poor practice about infection prevention of hepatitis B virus.