Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli ( E. coli) and Pichia pastoris ( P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients’ sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.