Background: Hepatitis B infection is a global threat for people of all age groups and is common in Bangladesh and other South Asian countries. Blood donors and blood donation can present important risk for hepatitis B infection. Our objective was to identify seroprevalence of hepatitis B virus among blood donors in Faridpur, Bangladesh. Methods: A cross-sectional study was conducted among 719 blood donors in the Faridpur district of Bangladesh from April to July 2013. Data was collected by non-probability convenient sampling maintaining inclusion and exclusion criteria. Face-to-face interview, blood grouping test, and hepatitis B surface antigen (HBsAg) screening were conducted. Results: About 30% of the blood donors were between 19 and 23 years of age, and 87.5% of them were males. Two-thirds (64.1%) of the donors were replacement donors, while the remaining 35.9% of them were voluntary donors. The overall seropositivity of HBsAg was found to be 1.5%. All infected blood donors were males. Hepatitis B infection was more prevalent in the 34–38 years age group and least prevalent among those older than 48 years of age. About 2% of replacement blood donors were infected with hepatitis B virus, compared to 0.7% among voluntary blood donors. Conclusion: Hepatitis B infection among blood donors is relatively lower than in other high-risk groups for hepatitis B infection in Bangladesh. Predominance of HBsAg among older adult groups, replacement donors, and the male gender can be a matter of public health concern.