In order to establish a rapid detection method for H7N9 avian influenza virus (AIV), monoclonal antibodies (mAbs) against hemagglutinin (HA) of H7N9 were developed to establish an antigen capture enzyme-linked immunosorbent assay (AC-ELISA). AC-ELISA achieved high specificity and sensitivity, with a detection limit of 3.9 ng/ml for H7N9 HA protein (A/Zhejiang/DTID-ZJU01/2013), and 2-2 HA unit/100 μl for live H7N9 AIV. The inter- and intra-assay coefficient of variation was less than 10%. Compared with conventional virus isolation detection, the sensitivity and specificity were 94.96% and 88.24%, respectively. AC-ELISA proved to be a rapid and practical technique for detection of H7N9 AIV.