Hepatitis C virus (HCV) infection has been noted a major public health problem with no vaccines available currently. This study aims at optimizing 'safe blood' practice by advancing HCV testing beyond Diaspot rapid enzyme immunoassay in current use in Nigerian Health Institutions. Between August, 2014 and November, 2015, a total of 300 blood donors' plasma samples were screened with both Diaspot and HCV Ag-Ab enzyme-linked immunosorbent assay techniques. HCV-RNA confirmation and quantification were performed with real-time polymerase chain reaction (PCR). Alanine aminotransferase (ALT) was performed on confirmed positive blood donor for treatment purpose. The overall gender ratio and mean age of blood donors screened for HCV were 1.5:1 and 27.67 ± 7.77 years respectively. Of the 300 blood donors screened, 5 (1.67%) and 1 (0.33%) were seropositive for HCV on the basis of Diaspot and enzyme-linked immunosorbent assay (ELISA) techniques respectively. Diagnostic odds ratio showed that ELISA is nearly 9-fold a better diagnostic tool compared to Diaspot technique. Real-time PCR assay confirmed positivity of 1 (0.33%) of the blood donor for hepatitis C. HCV-RNA viral load and plasma ALT of the lone sample were 133, 209 IU/mL and 11.1 IU/L respectively. HCV prevalence among blood donors in Ekiti state is low, 0.33%. Enzyme-linked immunosorbent assay technique should be the starting point of HCV serologic screening and a surrogate technique for real-time PCR. The development of workable algorithm to reduce risks associated with blood transfusion and enhances both blood donors' and recipients' safety is highly imperative.