Introduction: Human Immunodeficiency Virus (HIV) and syphilis co-infection is common and affects similar age groups. The presence of syphilis infection increases the chances of transmission of HIV. Syphilis has a negative impact on HIV infection, resulting in increasing viral loads and decreasing Cluster of Differentiation 4 (CD4) cell counts during syphilis infection. Aim: To determine the seroprevalence of syphilis, quantify HIV-Syphilis co-infection in patients attending an Integrated Counseling and Testing Centre (ICTC) centre. Materials and Methods: This cross-sectional study was conducted at the Department of Microbiology, Government Villupuram Medical College, Tamil Nadu, India, for a period of four months from March 2021 to June 2021. All consecutive adult patients attending ICTC during the study period for voluntary testing or referred from antenatal clinic or Sexually Transmitted Disease (STD) clinic were included. Patients who were on treatment or follow-up of syphilis were excluded. Pretest counselling session was given and consent was taken for HIV testing. The HIV antibody tests were performed and interpreted according to the National AIDS Control Programme (NACO) guidelines. Rapid Plasma Reagin test (RPR) screening was done. Positive cases were subjected to Treponema pallidum Haemagglutination Assay (TPHA) and Syphilis Rapid Immunochromatographic Test (RICT). The results were analysed with the data systematically entered in Microsoft Excel format and p-value was calculated by Fisher’s- exact test using epi infoTM software. Results: Among the 400 patients attending the ICTC who were included in the study, six patients had a reactive HIV test. The overall HIV positivity was 1.5% (6/400). The RPR test was positive in 7/400 patients (1.75%). Among the seven patients with RPR positivity, five patients were positive by TPHA and Syphilis RICT. Biological False Positivity (BFP) rate in RPR against TPHA was 28%. Thus the frequency of syphilis among the study population by the confirmatory test TPHA was 1.25%. Among the five confirmed cases of syphilis, four patients were males (80%) and one patient was female gender (20%). Four of the five patients were in the 28-37 year old age group (80%) and one patient was in the 38-47 year age group (20%). The frequency of syphilis was 16.67% among HIV positives (17%) and 1.02% among HIV negatives biological false positivity by RPR was observed in 29% of the cases. Conclusion: HIV and Syphilis co-infection is common and Specific Treponemal tests could contribute to reducing errors due to false positivity by non specific tests like RPR which can be used as a screening test in microbiology laboratories.