Avian influenza (AI) caused by H9N2 subtype avian influenza virus (AIV) poses a serious threat to poultry farming and public health due to the transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex (vRNP). It is of great significance for identifying the antigenic determinants of PB2 protein to explore the function of PB2 protein. In this study, PB2 sequence of H9N2 subtype AIV from 1090 to 1689 bp was cloned and expressed in the prokaryotic expression pET-28a vector. After purified, the recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the Hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting to PB2 protein were screened by indirect ELISA and Western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, 5H1) that stably secreted mAbs special to PB2 protein were screened, and heavy chain of 4B7 was IgG2α, that of 4D10 and 5H1 were IgG1, in which three mAbs were Kappa light chain. Also, the minimum B-cell epitope recognized by 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conservative among the different subtypes of AIV strains and located on the surface of PB2 protein. The above findings provide an experimental foundation for further investigation of the function of PB2 protein and provide effective technical support for developing monoclonal antibody-based diagnostic kits.