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Hepatitis B virus (HBV) infection is considered to be the ninth leading cause of death and is one of the major public health problems in the world. The prevalence of hepatitis in Nigeria has been rated to be 8.3%. Hence, this study investigate the prevalence of hepatitis B virus infection in a tertiary institution in Oyo State. Informed consent were obtained from the out-patients attending institution clinics. The design was a cross-sectional study and questionnaires were administered to investigate their knowledge and attitude towards safer practices against the spread of HBV. The seroprevalence of the participants were examined with LabAcon rapid kit with 98.2% - 100% and 97.2% - 99.8 for specificity and sensitivity respectively. The population distribution was observed to 45 (90%) female and 5 (10%) male. The prevalence of the HBsAg was found to be 10%. The prevalence of HBsAg among female participants was 11.1%. The relationship between knowledge of HBV infection and sex was insignificant (0.546) however the relationship between knowledge of HBV infection and age was significant (0.004). The occurrence of HBV infection is still area of concern because of the possibility of the virus to spread. This spread could better be curtailed based on regular awareness about the infection.