Background: Globally, the spread of HIV/AIDS remains on the rise especially among adolescents who are at increased risk of infection. Sexual behavioural change remains one of the most effective ways of preventing further transmission among this vulnerable group. Objective: To evaluate HIV/AIDS knowledge among undergraduate students' and how it can be used in HIV prevention strategies in Ghana. Methods: A cross-sectional study was conducted using structured questionnaires among 324 conveniently selected students enrolled at a tertiary institution in Accra, Ghana. Results: The mean age of the respondents was 23 years. Although the mean score of the participants' responses to 12 HIV/AIDS knowledge questions was 7.7 of 12 points, there was an inconsistent level of AIDS knowledge with significant gender difference. While students could identify the transmission modes and preventive measure, they were less knowledgeable about the causative agent of AIDS. Majority of the students reported having received AIDS information from both print and electronic media, but few of them received such information from parents. Although over 90% of the students knew where to access VCT services, 45% of them have not had HIV test. Conclusion: The data underscore the urgent need for HIV/AIDS-related health education and prevention efforts targeting university students as well as younger age groups in Ghana