Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. Influenza virus type A, which belongs to the family Orthomyxoviridae, is responsible for a serious infectious disease. H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals. Objectives: The purpose of the present experimental research was monoclonal antibodies (MAbs) production in contradiction of the NP of avian influenza virus (AIV) H9N2 subtype to detect AIV antigens and antibodies in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. in 2017. Methods: The conserved protein of NP from H9N2A/Chicken/Iran/259/2014 virus, with 60 kDa molecular weight, was isolated using the electroelution method. The purified protein was applied for Australian BALB/c mice immunization. After evaluating immunization by ELISA assay, the spleen of immunized mice was isolated and hybridized with SP2/0 myeloma cell. Next, the hybridized cell was cultured, and clone soups were collected after 15 days to examine antibodies via ELISA assay. The produced antibodies using Western blotting and antibody isotype kits were characterized. Results: Thirty antibody-producing clones were examined for reactivity against Nucleoprotein (NP), the antigen, at 1 µg/mL concentration. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen with titer 0.863 and 1.641, respectively. Two hybrid clones, 2D7 and 3F10, which produced anti-NP antibodies, were isolated and cultured. Characterizing of the produced antibodies using Western blotting was performed using H9N2 virus; finally, two clone soups (3/F10 and 2/D7) reacted with the NP virus protein. According to the isotyping of antibodies produced by 3F10 and 2D7 clones, 3F10 clone produced IgG1 with a κ chain, and IgG1 concentration was 1.997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a κ chain, and IgG2a concentration was obtained 1.951. Conclusions: According to the findings of our study, the produced antibody might be used in the diagnosis of influenza.