Environmental monitoring of avian influenza virus (AIV) in live poultry markets (LPMs) is vital for health risk forewarning, and survey of AIV environmental prevalence and biomolecular analysis of keystone subtypes remain essential. In this study, 970 biosamples were collected from air, poultry, water, and other environmental specimens of LPMs in Changsha from 2021 to 2023, and environmental prevalence survey of AIV and biomolecular analysis of H9N2 subtype were then conducted. Real-time fluorescent quantitative PCR was employed to detect the AIV and its subtypes (H5/H7/H9), revealing the positive rates of 68.45% for AIV and 53.51% for H9 subtype across all samples. Notably, aerosol samples had the positive rates of 89.00% for AIV and 72.50% for H9 subtype. Correlation analysis further revealed a negative correlation between the operation of exhaust facilities and the positive rates of AIV and H9 subtype. Subsequently, 30 AIV strains were isolated via chicken embryo inoculation, 12 of which were sequenced and identified to be H9N2 isolates. Genetic evolution analysis further indicated that these isolates were assigned into the G57 genotype. Furthermore, homology modeling revealed that mutations of V223A and N224H were identified at the receptor binding site of HA protein in several novel AIV isolates, which may lead to the variation in spatial conformation. Overall, the findings investigate the prevalence of AIV in LPMs and biomolecular attributes of H9N2 subtype, and provide some theoretical reference for future health risk assessment and preventive strategies of AIV.