Objective: To clone the hemagglutinin(HA) gene from H5N1 highly pathogenic avian influenza virus,and get eukaryotically expression. Methods: Total RNA was extracted from virus A/Jiangsu/08-6(H5N1),and used to reversely transcribe cDNA. HA gene was amplified with HA gene specific primers and then cloned into the pFastBac-GP67B plasmids with digestion by restriction enzyme BamHⅠ and XhoⅠ. The recombinant plasmid pFastBac-GP67B-HA was identified by restriction enzyme and gene sequencing. Following the transformation of E. coli DH10Bac competent cells with pFastBac-GP67B-HA,the recombinant bacmid rBacmid-HA was identified by blue/white selection and confirmed by PCR analysis. Then rBacmid-HA was transfected into sf9 cells using the Cellfectin Ⅱ reagent to eukaryotically express HA protein. The recombinant HA protein was characterized by SDS-PAGE,Western-blot and hemagglutination test and mass spectrographic analysis. Results: HA protein was efficiently expressed in sf9 cells with a molecular weight of about 70 000. In hemagglutination test,the recombinant protein HA can agglutinate chicken erythrocytes significantly. The mass spectrographic analysis confirmed the protein identity. Conclusion: The recombinant HA protein has the same biological activity as the natural HA protein,and the protein could be used to develop influenza specific subunit vaccine and functional antibody in future research.