Hepatitis C virus (HCV) infection in hemodialysis patients is mainly associated with insufficient precautionary measures. Compared with the general population, the prevalence of HCV infection is high among hemodialysis patients. HCV infection in these patients is typically associated with increased morbidity and mortality; however, the early detection of HCV may retard disease progression and reduce morbidity and mortality. In this study, we sought to estimate the seroprevalence of HCV infection among patients undergoing hemodialysis in a tertiary care hospital in Chennai, Tamil Nadu, South India. This was a prospective, observational, cross-sectional study that included 82 hemodialysis patients. Blood samples were collected from each patient, and the serum was separated for detection of the presence of HCV antibodies, HCV core antigen, and HCV RNA using an enzyme-linked immunosorbent assay, enzyme immunoassay, and polymerase chain reaction, respectively. Relevant socio-demographic, clinical, and laboratory information was also obtained. Among samples collected from the 82 patients, seven were found to be positive for HCV antibodies, whereas nine and eleven samples were found to be positive for HCV core antigen and HCV RNA, respectively. Long durations of renal failure and hemodialysis were identified as being positively associated with HCV infection in these hemodialysis patients, and a statistically significant reduction in hemoglobin levels and an increase in creatinine levels were detected among the HCV-positive patients. The overall seroprevalence of HCV infection in hemodialysis patients was found to be 13.4%. Given that high rate false-negative reactions were detected using ELISA and EIA, screening of HCV RNA during the early stages of HCV infection can contribute to preventing its nosocomial spread in these patients. Moreover, it is imperative that stringent universal precautionary measures are implemented in all health care settings and hemodialysis units to minimize the risk of HCV transmission.