During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.