To investigate the molecular epidemiology and genetic variation of H9N2 subtype avian influenza virus (AIV) in China from December 2023 to September 2024, and to screen for vaccine strains with improved immunogenicity, 280 clinical samples were collected from broilers and layers with suspected AIV infection across Gansu, Guangxi, Shandong, Hebei, Liaoning, Heilongjiang, Guangdong, Hubei, Jiangsu, Anhui, Henan, Inner Mongolia and Shanxi provinces. H9 subtype AIV nucleic acid detection, HA gene sequencing and virus isolation were performed. Using the isolated viruses, oil-emulsion inactivated vaccines were prepared and compared to commercial trivalent inactivated vaccines through cross-hemagglutination inhibition (HI) tests and challenge protection experiments to identify strains with superior immunogenicity. The results showed that 24 out of the 280 samples tested positive for H9N2 subtype AIV. All HA genes from these positive samples belonged to the h9.4.2.5 clade (also known as the Y280 lineage), with nucleotide homology ranging from 90.10% to 99.90% and amino acid homology from 91.44% to 99.82%. The HA protein cleavage site of all isolates was 333PSRSSR↓GLF341, consistent with low-pathogenic AIV characteristics. Key mutations observed at the receptor-binding site included R381K, Q234L, and T198V. Nine virus strains were isolated using specific pathogen-free (SPF) chicken embryos, among which A/Chicken/Shandong/JY17/2024 (H9N2) (designated SD17 strain) showed higher cross-HI antibody titers. The oil-emulsion inactivated vaccine prepared from the SD17 strain provided no less than 90% immune protection against circulating strains. The results indicate that the h9.4.2.5 clade is the predominant HA gene branch of H9 subtype AIV circulating in China. Combined with key amino acid site analyses, H9 AIV is gradually adapting toward mammalian hosts and aerosol transmission. The SD17 strain isolated in this study demonstrated excellent immune protection and can serve as a vaccine candidate for the development of new H9N2 subtype AIV vaccines.