Introduction: Blood donation is considered an important source of infection transmitted through transfusion, especially in developing countries like Pakistan., Objective: To find out the frequency of seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among blood donors in the blood bank., Methods: A prospective cohort study was carried out on blood donors at the National Institute of Blood Disease and Bone Marrow Transplant, Karachi, during the period of January 1, 2019 to December 31, 2020. The descriptive statistical analysis to find out the percentages and frequencies was implemented using SPSS version 23 (IBM Corp., Armonk, NY)., Results: During the study duration, a total of 23,656 blood donors visited and donated blood, including 12,234 blood donors in the year 2019 and 11,422 blood donors in the year 2020. According to the analysis, only 1.4% of patients with HBV, 1.5% with HCV, and 0.03% were seropositive in the year 2020. In 2019, 1.6% HBV, 2.07% HCV, and 0.09% HIV blood donors were seropositive with a significant 0.00 p -value., Conclusion: It is concluded that hepatitis C is the most commonly occurring in donors compared to HBV and HIV. HBV vaccines are available in Pakistan, which is why cases are fewer than HCV., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Ahmed et al.)