Abstract: This research aimed to study HA gene of H5N1 avian influenza virus pathogen from the perspective of molecular biology and explore its genetic variation and protein structure. Primer 5.0 was used to design a pair of primers according to the sequence of HA gene of H5N1 avian influenza virus published in GenBank. HA gene was amplified, cloned to pMD18-T vector and then identified. The recombinant plasmid pMD-AIV-HA, which was identified to be correct, was then analyzed by nucleotide sequence analysis. Bioinformatics software was used to do phylogenetic analysis of HA gene, amino acid sequence homology analysis, conserved sequence analysis, phosphorylation sites analysis, protein repeated sequence analysis and the secondary and tertiary structure prediction of HA protein. The sequencing results revealed that HA gene was 1661 bp and encoded 326 amino acids. The analysis of bioinformatics indicated that HA gene was conserved in species, and amino acid sequence homology between HA gene of H5N1 avian influenza virus and of other different AIV strains could reach 99.7%. It was also a highly pathogenic strain. The secondary and tertiary structure prediction results manifested that HA protein mostly consisted of α-helix, β-turn and random coil, and was a hydrophilic protein. The results would lay a foundation for the molecular virology research of AIV.