Background: Globally, more than 2 billion people have been infected with Hepatitis B Virus (HBV) at some time in their lives. Of these, about 350 million people remain infected chronically and become carriers of the virus. The main aim of this study was to assess the prevalence of HBV infection and associated factors among blood donors at Dire Dawa Blood Bank Service in Eastern Ethiopia. Methods and materials: This study involves cross-sectional retrospective record review on 4,157 blood donors. During analyzing the data, descriptive analyses was made to determine the prevalence of hepatitis B virus in the study population. Finally, multivariable logistic regression model was used to identify factors associated with hepatitis B virus infection. Result: Out of the 4,157 individuals who donated their blood at Dire Dawa Blood Bank Services, 155 (3.73%) (95% Confidence Interval (CI)=3.15-4.31) confirmed having a sero-prevalence positive for HBV. HBV seroprevalence has shown a statistical significance association with male sex (Adjusted Odds Ratio (AOR)=1.93, 95% CI=1.10-3.55) (p=0.036) and age group 33-40 (AOR=3.7, 95% CI=1.19-9.56) (p=0.029). Conclusion: The prevalence of HBV infection in this study is high and the disease is still a major health problem in the region which calls for high alert public health intervention.