Background: Hepatitis B virus is one of the transfusion transmission infectious agents of public health relevance. Its prevalence varies across the globe. Establishing the sero-prevalence of the disease is critical to informing the direction of preventive and control strategies. Objective: The aim of this study therefore was to establish the hepatitis B surface antigen sero-prevalence among blood donors in the Kintampo municipality of Ghana. Methodology: This three-year hospital based retrospective study was conducted at the laboratory unit of the Kintampo Municipal Hospital. The laboratory uses one step immunochromatographic test kits to detect the presence of hepatitis B surface antigen in the sera of blood donors. Records on blood donors were reviewed for hepatitis B sero-positivity. Results: A total of 3402 blood donors were studied, out of which 3238 (95.2%) were males and 164 (4.8%) were females. The overall sero-prevalence of hepatitis B surface antigen was 9.6% (327/3402). Majority of the sero-positive donors were less than 40 years, with higher prevalence of 16.4% (35/214) in donors less than 20 years. The sero-prevalence in males and females was 9.7% (313/3238) and 8.5% (14/164) respectively. Conclusion: The Kintampo municipality has a relatively high prevalence of hepatitis B among blood donors. The probability of hepatitis B viral infection was age inclined as the youth seem to be at greater risk of contracting the disease.