Introduction: Hepatitis B virus (HBV) is a severe liver infection with potentially fatal consequences. It ranks as the 10th leading cause of mortality globally and is closely linked to the development of liver cirrhosis and hepatocellular carcinoma. Consequently, the primary objective of this study is to determine the prevalence of HBV infection in tertiary health institutions in Bayelsa State, Nigeria. Methods: This cross-sectional study was conducted from September 2020 to January 2023 with September 2021 to June 2022 in Yenagoa. A total of 656 participants, comprising both males and females, were recruited for this research. Each participant’s HBsAg b virus status was assessed using a test strip. Results: Among the 656 participants screened, 10.1% tested positive for HBsAg, with the highest prevalence of HBsAg infections observed in participants aged 26 to 35, demonstrating statistical significance (P < 0.05, P = 0.097). Among the participants, females had a 5.5% infection rate with HBsAg B Virus, compared to their male counterparts, although this difference was not statistically significant (P > 0.05, P = 0.763). Conclusion: This study reveals the significant prevalence of HBV infection in two major tertiary health institutions in Bayelsa State. Consequently, it underscores the critical need for raising awareness and implementing rigorous public health policies to mitigate the risk of both vertical and horizontal transmission of the Hepatitis B virus.