Background: The percentage of HIV cases due to blood transfusion has decreased significantly in the last 2 decade. The newer 4th generation enzyme linked immunosorbent assay (ELISA) has been shown to have increased detection rate compared to 3rd generation ELISA. Objectives: To estimate the seroprevalence of HIV among blood donors using 4th generation ELISA assay and to compare it with the 3rd generation ELISA. Materials and Methods: This retrospective and prospective study involved 32,360 blood donors- 25,420 were voluntary donors and 6,940 were replacement donors. All blood units were tested with 3rd as well as 4th generation ELISA. All samples found reactive or in grey zone with either 3rd or 4th generation ELISA were retested by re-ELISA & Rapid HIV Card Test. Results: The seroprevalence of HIV was estimated to be 0.55/1000 donations (0.05%) with 3rd generation ELISA compared to 0.89/1000 donations (0.09%) with 4th generation ELISA. The seroprevalence of HIV among voluntary donors was estimated to be 0.47/1000 donations (0.04%) with 3rd generation ELISA and 0.74/1000 donations (0.07%) with 4th generation ELISA. The prevalence of HIV among replacement donors was 0.86/1000 donations (0.08%) with 3rd generation ELISA and 1.44/1000 donations (0.14%) with 4th generation ELISA. Conclusion: 4th generation HIV ELISA detects a higher number of seroreactive donors compared to 3rd generation ELISA. However, larger studies are required with confirmatory tests for both 3rd and 4th generation ELISA for making any policy changes.