To estimate the prevalence of Human immunodeficiency virus [HIV], Hepatitis B virus [HBV] and Hepatitis C virus[HCV] among patients attending a tertiary care hospital, South India. A cross-sectional study was conducted between November 2015 and August 2016. A total of 71,703 samples [HIV-319, HBV-455, HCV- 157] were tested for anti-HIV, HBS Ag and anti-HCV using standard serological tests. The prevalence of HIV was 319[1.44%], HBV 455 [1.78%] and HCV was 157[0.65%]. ‘Z’ test was used for statistical analysis. It was found that there was statistically significant difference between proportions of all the three. The proportion of HBV was more when compared with HIV [P=0.0019] and HCV [P=0.00001]. The proportion of HIV was more when compared with HCV [P=0.00001]. The study revealed high prevalence of HBV, intermediate prevalence of HIV and low prevalence of HCV. The study emphasises the need for early screening of all the three infections, and the results highlight the importance of relevant targeted interventions in these populations.