Objective: To analyze the genomic characteristics of human infection with H9N2 avian influenza virus in Gansu province. Methods: The etiological analysis was conducted for human infection with H9N2 avian influenza virus detected in influenza like illness cases in northwestern China in 2016. Molecular bioinformatics Mega 7.0 software was used to analyze the full genomic sequences of the viral isolate. Results: The gene fragments of HA, NA, MP, NP, NS, PA, PB1 and PB2 of the isolate were highly similar (>90%) to those of H9N2 avian influenza virus strain isolated in external environment in Gansu from 2014 to 2019. The HA gene belonged to BJ/94-like branch, PB2 and MP belonged to G1/97-like branch, and the PB1, PA, NS, and NP genes belonged to F/98-like branch. MP and PB2 were closely related to H7N9, H10N8 and H5N6 viruses. Amino acid sequence alignment showed that the HA cleavage site was arranged in PSRSSR ↓ GLF, H183N and Q226L mutated which included 7 HA glycosylated sites; 62-64 sites of NA absented 3 amino acids (ITE); and M2-31N, NS1-42S, PA-356R, and PA-409N mutated. Conclusions: Apparently, this case of human infection with human infection with H9N2 avian influenza virus was an incidental. However, the isolates of H9N2 influenza virus in external environment of Gansu had a series of mammalian adaptive molecular markers, suggesting that the risk of human infection is higher. It is necessary to strengthen the surveillance by multi departments to deal with influenza pandemic.