ABSTRACTHepatitis C virus is one of the emerging infectious diseases that can be transmitted through blood-to-blood contact. This study was carried out to determine the prevalence of anti-HCV antibodies among potential blood donors and pregnant women attending Bowen University Teaching Hospital (BUTH), Ogbomoso, Oyo State. This hospital-based study was conducted from December 2014 to September 2015. The study group (N = 279) included potential blood donors and pregnant women. Data on socio-demographic characteristics and potential risk factors were collected using a structured questionnaire. The presence of anti-HCV antibodies in serum samples of the studied subjects were detected using third-generation Enzyme Linked Immunosorbent Assay (ELISA) (WKEA Med Supplies Corp, China). Chisquare test was utilized to assess the association between the socio-demographic variables and HCV status. Logistic regression was done to determine the strength of association between risk factors and HCV status. Statistical significance was set at P ˂ 0.05. Overall seroprevalence of hepatitis C virus infection was found to be 1.79% consisting 0.36% of pregnant women and 1.43% of blood donors. None of the socio-demographic characteristics and potential risk factors among the study groups were significantly associated with hepatitis C virus infection. This study found a seroprevalence of anti-HCV antibody to be 1.79%, thus, screening of pregnant women and blood donors for HCV infections with the use of ELISA is recommended because of its important role in detecting the presence of anti-HCV antibody with utmost specificity and sensitivity.