Context:The ultimate goal of a blood transfusion service is the provision of safe and adequate supply free from transfusion-transmitted infections (TTIs). TTIs not only threaten the recipient's safety, but they also increase disease burden. Seroprevalence of TTIs in healthy blood donors indirectly reflects the prevalence of these infections in the general healthy population.Aim and Objectives:To study the seroprevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) in healthy donors at a tertiary care hospital-based blood bank. To know the yearly and age-group prevalence of these TTIs as compared with other studies across India.Settings and Design:This is 5 years observational cross-section study conducted in a tertiary hospital-based teaching institute of Central India (Malwa region).Materials and Methods:The results of serological testing of TTIs and donor variables were analyzed during 2011–2015.Statistical Analysis Used:Chi-square test and Chi-square for trend analysis on TTIs prevalence.Results:A total of 58,998 donors were screened for TTIs with dominance of male donation (99.7%). The overall cumulative seroprevalence was 1.14% in our study. The seroprevalence of HIV, HBV, and HCV was 0.09%, 0.98%, and 0.07%, respectively. We found a statistically significant increasing trend for HCV seropositivity during the study.Conclusion:Our study reflects an increasing trend of HCV seroprevalence over time. Thus, efforts are needed to increase the awareness and to educate the population in reducing risk factors for HCV infection.