Hepatitis B virus infection represent a major public health problem responsible for acute and chronic viral infections, HBV-related liver disease or hepatocellular carcinoma. Sub-Saharan Africa is worse hit by HBV infection and its related morbidity and mortality. Despite the burden of HBV in the region, the challenges confronting people living with HBV include making diagnosis and provision of their comparable estimates. This study aimed at analyzing the sero-prevalence of HBsAg among health workers and possible challenges in assessment of HBV prevalence in a resource-limited sub-Saharan African country. Blood samples from 275 consented health workers were tested for HBsAg using third generation ELISA. Data analysis was obtained using SPSS version 20. HBsAg screening was performed on a consecutive sample of 275 respondents (95 males and 180 females with M/F ratio of 1:1.9) aged 18-59 years who voluntarily turned up for the survey. Mean age ± SD was 36.1 ± 9.6 years. The overall prevalence of HBV was 1.5% (4/275). The prevalence in females 3/180 (1.7%) was insignificantly higher than males 1/95 (1.1%) (P>0.05).In relation to age group, the younger age group (< 20 years) has significantly higher prevalence (1/5 (20%) compared to other age groups (P=0.02). Blood transfusion and vaginal discharge (STD) were the highest predisposing factors to HBV infection with ORs of 5.9 and 4.2 respectively. This study, although limited by small sample size, showed a reduced prevalence of HBV among the health workers. The result from this study may not be a true reflection of the prevalence of HBV in south-eastern Nigeria, hence the need to involve larger population size in subsequent study. There is need to institute elaborated serological and virological diagnostic markers for the diagnosis of HBV infection in our health institutions. Also, targeted group health education and national guidelines for hepatitis B prevention and treatment must be provided by the Federal Ministry of Health, Nigeria. These interventions will go a long way to curb the prevalence of HBV infection to the barest minimum.