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 (HCWs) in Kisumu County. We determined prevalence of HBV infections among 192 HCWs from nine purposively selected high-patient 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). Of 192 HCWs sampled, 52.1% were males and the median participants age was 34.4 years with interquartile range (IQR) of 11 (28–39) years. Most participants (44%) had worked for between 1–5 years. There was low HBV vaccine uptake with 35.9% completing the required 3 doses, while 40.6% had never been vaccinated. HBV prevalence was 18.8% (36/192), prevalence of past resolved infection was 25.5% (49/192), while 37.5% (72/192) of HCW had evidence of vaccine-derived immunity and 17.7% (34/192) were susceptible. HBV prevalence among HCW who had worked for less than one year and those who had never been vaccinated was 37.5% and 35.9% respectively. Significant risk of HBV lifetime exposure was noted among HCWs with one vaccine dose, those with no known exposure, while highest in those with knowledge on HBV transmission (aOR, 7.97; 95% CI, 2.10–153.3, p-value = 0.008). HCWs 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 HCWs. HBV prevalence was high among HCWs from nine high patient volume public hospitals in Kisumu County. Efforts to strengthen HBV vaccination uptake and dose completion are needed to reduce HBV infections among HCWs.