Background. Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV) and major global health problem, affecting millions of people globally. Whilst college or university students could serve as a positive vehicle that may aid in the propagation of education in the communities, there is currently paucity of data on knowledge of HBV and vaccination awareness among university students in Ghana. This study therefore assessed knowledge on hepatitis B virus infection, testing, and vaccination awareness among science and non-science students in Ghana. Method. This cross-sectional study included 386 students from the Garden City University College, Kumasi, Ghana, from February to June 2022. A well-structured questionnaire was used to obtain data on knowledge, testing, HBV vaccination status, and sociodemographic characteristics of participants. All statistical analyses were done using SPSS Version 26.0 and GraphPad prism version 8.0. A Chi-square test statistic and logistics regression analyses were used to determine factors associated with study variables among study participants. p value of <0.05 and 95% confidence interval were considered statistically significant. Results. About half (51.5%) of the participants had adequate knowledge on hepatitis B infection with 22.3% demonstrating poor knowledge on hepatitis B infection. A little more than average (51.6%) previously tested for hepatitis B (HBV) whilst 32.9% were highly aware of hepatitis B vaccination and 33.9% were not aware of hepatitis B vaccination. This study found that ethnicity of students (p=0.0020), family history of hepatitis B infection (p=0.0160), and academic cumulative weighted average (CWA) (p=0.0020) were significantly associated with knowledge about hepatitis B infection. Also, students reading science-related programs had more than twice the odds (aOR = 2.56, 95% CI (1.03–5.08), p=0.0210) of having tested for HBV infections before compared to students who read non-science programs. Furthermore, sex (p<0.0001), family history of HBV vaccination (p=0.0260), CWA (p=0.0060), and the program of students (p=0.0020) were significantly associated with awareness of HBV vaccination. Conclusion. Knowledge of HBV infection among university students is satisfactory but awareness of HBV vaccination and testing is poor. There is a need to enhance educational interventions to improve the general knowledge of HBV infection, testing, and vaccination in Ghana especially among non-science students.