Many cases of avian influenza A(H7N9) virus infection in humans have been reported since its first emergence in 2013. The disease is of concern because most patients have become severely ill with roughly 30% mortality rate. Because the threat in public health caused by H7N9 virus remains high, advance preparedness is essentially needed. In this study, the recombinant H7N9 hemagglutinin (HA) was expressed in insect cells and purified for generation of two monoclonal antibodies, named F3-2 and 1C6B. F3-2 can only recognize the H7N9 HA without having cross-reactivity with HA proteins of H1N1, H3N2, H5N1, and H7N7. 1C6B has the similar specificity with F3-2, but 1C6B can also bind to H7N7 HA. The binding epitope of F3-2 is mainly located in the region of H7N9 HA(299-307). The binding epitope of 1C6B is located in the region of H7N9 HA(489-506). F3-2 and 1C6B could not effectively inhibit the hemagglutination activity of H7N9 HA. However, F3-2 can prevent H7N9 HA from trypsin cleavage and can bind to H7N9 HA which has undergone pH-induced conformational change. F3-2 also has the ability of binding to H7N9 viral particles and inhibiting H7N9 virus infection to MDCK cells with the IC50 value of 22.18 mu g/mL. In addition, F3-2 and 1C6B were utilized for comprising a lateral flow immunochromatographic test strip for specific detection of H7N9 HA.