Hemagglutinin (HA) gene is an important gene of protective antigen of Avian Influenza Virus (AIV).In order to study HA gene vaccine, the H5 gene of AIV, amplified by PCR, was subcloned into eukaryotic expressing vectors pcDNA4/HisMax and pRc/CMV. The recombinant plasmids were transfected into HeLa cell by Tfx^TM-20 transfection reagent, Superfect transfection reagent and electroporation respectively. The expressed HA was examined and identified by Western blotting and hemagglutination assay. The results showed that HA was expressed precisely in HeLa cells and had bioactivity. Three special bands (HA, HA1 and HA2) were found by Western blotting, which were identical to those of AIV. The quantity of expressed HA from pC4H5,transfected by superfect transfection reagent, was approximately 8 times as much as that from pCMVH5. These results suggest that pC4H5 is a highly efficient eukaryotic expressing vector.