Abstract: Indirect enzymelinked immunosorbent assay (ELISA) for detecting antibodies against avian influenza virus (AIV) was developed by using expressed full length nucleoprotein (NP) of H9N2 AIV in E.coli. 263 chicken serum samples (including 243 clinical serum samples and 20 positive serum samples from H9N2 AIV vaccinated chicks) were detected by NP-ELISA, agar gel precipitin test (AGP), and hemagglutination inhibition (HI). The results showed that the coincidence ratio between NP-ELISA and AGP or HI was 83.3% and 92% respectively. The specific assay suggested that NP-ELISA was able to detect H5，H7 and H9 subtype antibodies to AIV, and the serum samples which were confirmed positive by NP-ELISA could be blocked by positive chickenembryo allantoic fluid. The sensitive analysis demonstrated that NP-ELISA can detect specific antibody in the 7th day after AIV infection in chicks, and all sera were positive in the 10th day. However, serum samples were still negative at the 21st day post inoculation (PI) by AGP test, and HI tests began to detect low levels of antibodies at the 10th day PI. The sensitivity of NP-ELISA was 4-40 times higher than that of HI . The present study confirmed that the NP-ELISA was a rapid, sensitive, economic and specific method for typeserologically detection of AIV infection in chickens.