Background: Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) are significant public health concerns globally, particularly in sub-Saharan Africa, where the burden of these infections is disproportionately high. Blood donors play a critical role in ensuring a safe blood supply, yet there is limited data on the prevalence of these infections among blood donors in Ghana. This study aimed to assess the prevalence of HIV and Hepatitis B among blood donors at the Greater Accra Regional Hospital and to evaluate their awareness of these infections. Methodology: This analytical cross-sectional study was conducted at the Greater Accra Regional Hospital. A total of 100 voluntary and replacement blood donors were selected using a convenience sampling method. Data were collected using a structured questionnaire, which assessed participants' socio-demographic characteristics and their awareness of HIV and Hepatitis B. Blood samples were tested for HIV and Hepatitis B using the Enzyme-Linked Immunosorbent Assay (ELISA). Descriptive statistics, including frequencies and percentages, were used to summarize the data. Results: The prevalence of HIV among the blood donors was 2%, with 98% testing negative. The prevalence of Hepatitis B was 1%, with 99% of donors testing negative. Awareness levels for HIV and Hepatitis B were high, with 98% of donors aware of HIV and 99% aware of Hepatitis B. However, there were gaps in knowledge, particularly regarding Hepatitis B prevention and the risks of co-infection with HIV. Conclusion: The study found a low prevalence of HIV and Hepatitis B among blood donors at the Greater Accra Regional Hospital, indicating a relatively safe blood supply. However, gaps in knowledge about Hepatitis B and co-infection risks highlight the need for continued public health education. Strengthening screening protocols and addressing knowledge gaps regarding prevention, particularly Hepatitis B vaccination, are essential to ensure the safety of the blood supply in Ghana and sub-Saharan Africa.