OBJECTIVE Enzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein. METHODS rHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice. RESULTS Our ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model. CONCLUSIONS An ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.