BACKGROUND : Human immunodeficiency virus, hepatitis B virus and hepatitis C virus are among the greatest threats to blood safety for the recipient. They are also the leading cause of death, chronic and life-threatening abnormalities. Therefore, this study was aimed to assess the Sero-prevalence of HIV, Hepatitis B and C virus among blood donors at the University of Gondar Comprehensive Specialized Hospital. METHODS : A retrospective cross-sectional study was used to estimate the seroprevalence of HIV, Hepatitis B and C virus among blood donors at the University of Gondar Comprehensive Specialized Hospital from May-July 2018. Screening of HIV, HBV, and HCV was done by using the Enzyme-Linked ImmunoSorbent Assay. Records of 5983 first time blood donors were collected and reviewed by using a checklist from registration book. Data was entered in statistical package EP Info version 3.5.1, and data cleaned and analyzed using the statistical package SPSS version 16.0. RESULTS : Of 5983 blood donors, 85.5% (5118/5983) donors were males and 14.5% (865/5983) were females. The median age was 27 years and the highest blood donations age category was between 20 to 51.2% (29/5983) followed by 30 to 39 years of age, 21.6% (1295/5983). The prevalence of HIV, HBV and HCV infections were 2.5% (95% CI: 1.07-2.398), 4.1% (95% CI: 0.461-1.053) and 1.6% (95% CI: 0.845-3.354), respectively. HIV infection was significantly associated with gender (p = 0.021, x2 = 5.358) and HCV infection with age group (p = 0.003, x2 = 17.673). Of all donated blood, 8.2% (489/5983) had serological evidence for at least one of the screened pathogens and 58 (0.96%) of them had multiple infections. CONCLUSIONS : This study showed a significant prevalence of HIV, HBV, and HCV among blood donors, 2.5% (147/5983), 4.1% (244/5983) and 1.6% (98/5983), respectively. Therefore, strict selection of blood donors with an emphasis on getting voluntary blood donors, and highly sensitive and specific tests for screening of blood donors for HIV, HBV, and HCV using standard methods are highly recommended to ensure the safety of blood for the recipient.