The H9N2 avian influenza virus (AIV) remains a serious threat to the global poultry industry and public health. The hemagglutinin (HA) protein is an essential protective antigen of AIVs and a major target of neutralizing antibodies and vaccines. Therefore, in this study, we used rice-derived HA protein as an immunogen to generate monoclonal antibodies (mAbs) and screened them using an immunoperoxidase monolayer assay and indirect enzyme-linked immunosorbent assay. Eight mAbs reacted well with the recombinant H9N2 AIV and HA protein, four of which exhibited potent inhibitory activity against hemagglutination, while three showed remarkable neutralization capacities. Western blotting confirmed that two mAbs bound to the HA protein. Linear epitopes were identified using the mAbs; a novel linear epitope, 480HKCDDQCM487, was identified. Structural analysis revealed that the novel linear epitope is located at the C-terminus of HA2 near the disulfide bond-linked HA1 and HA2. Alignment of the amino acid sequences showed that the epitope was highly conserved among multiple H9N2 AIV strains. The results of this study provide novel insights for refining vaccine and diagnostic strategies and expand our understanding of the immune response against AIV.