Infection with hepatitis C virus (HCV) is a major cause of transfusion-associated hepatitis, cirrhosis and hepatocellular carcinoma. The present study was conducted with an objective to evaluate the prevalence of anti-HCV antibody in New Delhi, India using a large number of healthy voluntary blood donors. A total of 15,898 healthy voluntary blood donors were subjected to anti-HCV testing (using a commercially available third generation anti-HCV ELISA kit) and 249 were found to be reactive for anti-HCV antibody, yielding an overall prevalence of 1.57%. No significant difference was found between the HCV positivity rate of male (1.57%; 238/15,152) vs. female (1.47%; 11/746) donors, family (1.58%; 213/13,521) vs. altruistic (1.51%; 36/2377) donors and first-time (1.55%; 180/11,605) vs. repeat (1.61%; 69/4293) donors. The age distribution of anti-HCV reactivity showed a maximum prevalence rate of 1.8% in the age group of 20–29 years. In addition, there was a clear trend of decreasing positivity for anti-HCV with increasing age and this trend was statistically significant. The results of the present study show that the prevalence of anti-HCV antibodies in the healthy voluntary blood donors of New Delhi, India is considerably higher than the reported seroprevalence of HCV in majority of the industrialized nations and this represents a large reservoir of infection capable of inflicting significant disease burden on the society. In addition, donors of New Delhi, India showed a trend of decreasing seroprevalence with increasing age, possibly implying a higher exposure rate to HCV in younger subjects.