Introduction: Human Immunodeficiency Virus (HIV) affects the immune system causing body’s normal defense to break down, leaving it vulnerable to a host of life threatening diseases. High risk behaviors and routes of transmission for HBV and HCV are identical with HIV and other sexually transmitted diseases (STDs). Hence this study is carried out to determine coinfection of Hepatitis B, Hepatitis C and Syphilis among HIV positive patients on Antiretroviral Therapy at Regional STI Training, Research and Reference Laboratory (RSTRRL), Department of Microbiology, Mumbai. Aim and Objective: To determine seroprevalence of Hepatitis B, Hepatitis C and Syphilis coinfection in HIV patients on antiretroviral therapy. Material and Methods: A retrospective study was conducted during April 2016 to March 2018 in RSTRRL. Blood samples received in laboratory from ART clinic for HBV, HCV and syphilis testing were included in study. HBsAg and HCV antibody ELISA and VDRL Calcutta antigen for syphilis were performed. All VDRL positive samples were also tested by TPHA. Results: 2771 patients on ART attending ART centre were included in this study. Maximum patients were males(55.18%). 66.73% of these were in the age group of 26–45 years which is the reproductive age group. Seroprevalence of Hepatitis B, Hepatitis C and syphilis was 4.3%, 1.6% and 1.8% respectively. Co–infection of HBV and syphilis, HBV and HCV, HCV and syphilis were found to be 0.4%, 0.07% and 0.04% respectively. Conclusion: Screening of Hepatitis B, Hepatitis C and syphilis should be included in diagnostic algorithm of HIV positive patients on ART as the antiretroviral drugs could be hepatotoxic. Hepatitis B screening and Hepatitis B vaccination should be given to HBsAg negative individuals attending ICTC should be initiated in the ICTC’s