The unprecedented spread of highly pathogenic avian influenza (HPAI) subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (mAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture ELISA (AC-ELISA) to detect H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1), expressed in bacteria or with concentrated H5N2 virus, yielded a panel of hybridomas secreting mAbs specific for influenza HA. The reactivity of each mAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, secreting IgG and IgM respectively, were used as the capture antibody and pooled hyperimmune guinea pig serum IgG served as detector antibody. The specificity of the optimized AC-ELISA was evaluated using AIV H5 H3, H4, H7, H9 and H10 subtypes. Specimens containing AIV H5 subtypes yielded a specific and strong signal above background, whereas all other subtypes yielded background signals. The detection limit of the AC-ELISA was 62.5 ng of bacteria-expressed H5N1 HA1 protein and 124, 62 and 31 TCID50 of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with normal chicken pharyngeal-tracheal mucus also yielded positive signals in the AC-ELISA and were confirmed by RT-PCR. The tracheal swab samples from H9N2 infected chickens did not give positive signals. Taken together, the newly developed mAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for specific detection of H5 avian influenza virus.