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Blood transfusion service comes with its various risks which include transfusion of transmissible infections especially hepatitis B. Though Nigeria is noted as a high endemic region for hepatitis B virus (HBV) infection, yet most techniques focus on detection of only hepatitis B surface antigen marker in serum or plasma. This study aims at optimizing ‘safe blood’ practice by advancing hepatitis B virus testing in prospective blood donors (PBD) beyond single marker serologic screening and estimating HBV endemicity among blood donors based on HBV markers seroprevalence. Up to 4 ml of K3EDTA anticoagulated sample was obtained from PBD and aliquot into two separate plain containers following informed consent and ethical approval. A total of four hundred and seventy (470) of PBD were initially screened for HBV markers using NOVA HBV 5-in-1 rapid one-step enzyme immunoassay between August, 2014 and November, 2015. Results were analyzed using SPSS version 21. The results showed that the overall gender ratio and mean age of PBD screened for HBV are 1.45:1 and 26.87±7.51 respectively. Chi square revealed right knowledge of most of the routes of hepatitis B viral transmission by PBD (χ2 range = 11.6 – 102.3, p < 0.05). Also, this study revealed HBsAg seroprevalence of 6.4% based on NOVA 5-in-1 rapid EIA. Cummulative HBV markers seroprevalence was 19.36% including 34 (7.23%) HBsAb+ and 1(0.21%) HBsAb+HBeAb+HBcAb+ showing evidence of vaccinated and naturally immunized donors respectively. In conclusion, the use of more stringent serologic techniques and workable algorithm to reduce risks associated with blood transfusion and enhance both blood donors’ and recipients’ safety is no longer a luxury but a necessity.