In this study, one H9N2 subtype avian influenza virus (AIV) was isolated in Jiangsu Province, China, in March 2025. According to phylogenetic analysis, the HA gene fragment of the H9N2 isolate was classified as the B4.7 lineage, with the remaining seven segments falling into the Eurasian lineage. This H9N2 virus underwent complex genetic reassortment across multiple regions in China during its evolution, and key amino acid substitutions were identified, which may enhance its viral polymerase activity and replication efficiency in mice. Critically, a mouse experiment demonstrated that the H9N2 virus replicated effectively in the lungs and nasal turbinates of BALB/c mice without requiring prior adaptation. In addition, poultry infection studies showed that the H9N2 virus could replicate and spread more efficiently in chickens than in ducks. Moreover, the virus could be transmitted to contacted ducks through infected chickens with greater efficiency than from ducks to ducks. Furthermore, in the lungs of inoculated chickens and ducks, the mRNA expression levels of pattern-recognition receptors (PRRs) and cytokines were upregulated, while the overall fold increase in ducks was lower than that in chickens. In conclusion, these results highlighted the potential public health risk presented by chicken-derived H9N2 AIV, emphasizing the critical need for enhanced routine surveillance of H9N2 AIVs from poultry.