Background: Avian influenza viruses (AIVs) including the subtype H9N2 cause considerable financial losses to poultry industries. Rapid and accurate diagnosis of avian influenza (AI) infection is important in control and eradication programs. OBJECTIVES: The aim of this study was to produce monoclonal antibodies (MAbs) specific for the nucleocapsid protein )NP (of AIV H9N2 subtype to improve diagnostic assays. METHODS: Recombinant NP protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen for mice immunization. Spleen cells of the immunized mice were fused with SP2/0 myeloma cells. Next, culture supernatants of primary hybridoma clones were screened by indirect ELISA. After three rounds of sub cloning, the reactivity of the MAbs with recombinant and natural antigens was assessed by Western blotting. RESULTS: Six MAbs showed specific binding to recombinant and natural NP from AIV H9N2 in Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay. Cross-reactivity with genetically non-related including Newcastle viruse (Paramyxoviridae family) was not detected. CONCLUSIONS: Based on the results, the MAbs generated in this study could be used for the development of rapid diagnostic assays for recognition of AIV.