BACKGROUND Hepatitis B vaccination among healthcare workers (HCWs) in Ghana has not been actively pursued despite the endemicity of the infection. This study measures the hepatitis B vaccine uptake among HCWs at the University of Ghana Hospital, Legon (UGHL) and identifies the factors associated with vaccination. METHODS An analytical cross-sectional study involving all staff who have direct contact with patients was conducted. Self-administered questionnaires were used to collect data on vaccination status, age, sex, type of staff, duration of work in the facility, exposure to blood or blood products, blood stained linens/waste, sharp instruments and performance of invasive procedures. Data was analysed using STATA 14. Continuous variables were described using median values and interquartile ranges (IQR) and categorical variables as proportions. Bivariate and multivariate analysis were conducted to identify the factors associated with hepatitis B vaccination status. RESULTS Of the 161 participants interviewed, 63.4% were females with median age 35 years (IQR: 27-45). Eighty-six (53.4%) of the respondents had taken the hepatitis B vaccine with 79.1% of them having completed the vaccination schedule. Factors associated with vaccination were working for more than 16 years (OR: 3.8, CI: 1.02-12.72), daily exposure to blood/blood products (OR: 4.1, CI: 1.43-11.81) and sharp instruments (OR: 4.45, CI: 1.39- 14.24), performing invasive procedures daily (OR: 3.0, CI: 1.07-8.45) and frequent exposure to blood stained linens/waste (OR: 6.1, CI: 1.41-26.51). CONCLUSIONS The lack of hepatitis B vaccination among some HCWs at UGHL puts them at risk of contracting hepatitis B infection.