Background Poorly managed medical waste produced at the health facilities are potential source of infections including occupational exposure to Hepatitis B Virus (HBV). This study evaluated the prevalence of HBV infection among healthcare workers (HCW) in Kisumu County. Methods We determined the prevalence of HBV infections among 192 HCW from nine purposively selected high volume public hospitals in Kisumu County. A structured questionnaire was administered, and 4.0 ml of venous blood sample collected for Hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs) and total hepatitis B core antibody (anti-HBc) testing using enzyme immunoassay (EIA). Results Of the 192 HCW sampled, 52.1% were males and 78.7% are married, the median participants age was 34.4 years with interquartile range (IQR) of 11 years. Most participants had between 1-5 years of service while 43.8% had >=2 doses of HBV vaccine. The respective prevalence of HBsAg, anti HBs and anti HBc was 18.8% (95% CI: 13.5-25.0%), 63.0% (95% CI: 55.8-69.9%) and 44.8% (95% CI: 37.6-52.1%). Higher proportion of HBV positive was found in HCW who had worked for less than one year and who had not received any dose of HBV vaccine at 37.5% and 35.9% respectively.. Significant risk of HBV lifetime ex­posure was noted among HCW with one vaccine dose, those with no known exposure and highest in those with knowledge on HBV transmission (aOR, 7.97; 95% CI, 2.10-153.3, p-value=0.008). HCW who had received >=2 doses of HBV vaccine (aOR, 0.03; 95% CI, 0.01-0.10, p-value= <0.0001) had significant HBV protection. Duration of service was not associated with HBV among HCW. Conclusion Prevalence of HBV infection was high among HCW in Kisumu County . Ministry of health Kenya should strengthen comprehensive infection prevention and control practices to reduce lifetime exposure to HBV infection among HCW.