The objective of present research was to establish antigen capture ELISA detection method for H9 subtype avian influenza virus. BALB/c mice were immunized with HA recombination protein, and then the mouse splenic cells were fused with SP2/0 cells, hybridoma cell stably secreting anti-HA McAb was screened by ELISA coated with the AIV antigen, an antigen capture ELISA based on monoclonal antibody was developed for detection of H9 AIV. Hybridoma cell stably secreting anti-HA McAb designated 5F10, the immunoglobulin type was IgG2b type with κ chain. The H9 AIV AC-ELISA showed no cross-reaction with other five avian viruses (ILTV, NDV, EDSV, IBV, and IBDV), the sensitivity eight times higher than HA test, Comparing with RT-PCR, the concordance was 94.8 %, the sensitivity was 97.1 %, and the specificity was 94.4 %. The H9 AIV AC-ELISA has good specificity, sensitivity and repeatability, could be used for diagnosing H9 subtype AIV infections.