Background. Hepatitis B virus (HBV) infection is a serious public health problem in many parts of the world. The risk of acquiring the infection through exposure to blood, semen, and other bodily fluids is highest among health care workers (HCW) including trainees. Ghana is considered a high risk country for HBV; however little is known about the knowledge and prevention practices of the infection in the country. This study assessed the knowledge, testing, and vaccination history of HBV and their related factors among undergraduate public health students of University of Health and Allied Sciences in Ghana. Methods. A cross-sectional study was conducted among 226 students using a pretested questionnaire to assess Hepatitis B knowledge, testing, and vaccination history of the students. We performed logistic regression analysis to examine the relationship between Hepatitis B testing and vaccination history and participants’ characteristics. Data was analysed using Stata Version 12. Results. Majority 169 (73.9%) of the 226 participants studied had moderate knowledge regarding HBV infection. About half 114 (50.4%) of them had never been tested for HBV infection, and 100 (44.2%) had received at least a single dose of Hepatitis B vaccine. The completed vaccination rate among the students was 30.5%. Students in their 2nd year (Adjusted Odds Ratio [AOR]: 3.13; 95% Confidence Interval [CI]: 1.13, 7.52; p<0.011) and those with moderate (AOR: 4.76; 95% CI; 1.35, 16.82; P=0.015) and good (AOR: 5.40; 95% CI: 1.31, 22.36; P=0.020) level of knowledge were more likely to be tested for HBV. With regard to vaccination, females (AOR: 1.85; 95%CI: 1.04-3.29; P=0.037) and regular students (AOR: 0.37; 95%CI: 0.19, 0.70; p=0.002) were associated with receiving the full dose of Hepatitis B vaccine. Conclusion. This study highlights the urgent need for continued health education on HBV infection and strategies that ensure that health trainees are screened and fully vaccinated against the infection to prevent potential future exposure to the virus. The students’ representative council can organize free HBV testing and vaccination for all fresh students.