Objective:To construct the full-length NS1 gene of influenza A(H7N9) into an eukaryotic expression vector PXJ40-MYC,and study the expression of NS1 gene in transfected 293T cell. Methods:The NS1 gene of influenza A (H7N9) was amplified by RT-PCR and cloned into pMD18-T vector to construct a plasmid,named pMD18-T-NS1.The PCR product of pMD18-T-NS1 plasmid and the PXJ40-MYC were double digested using the same restrict enzymes,the recombinant eukaryotic expression vector PXJ40-MYC-NS1 was subsequently yielded. The expression of the NS1 gene in transfected 293T cells was tested by Western blot. Results:The recombinant eukaryotic expression vector PXJ40-MYC-NS1 was successfully constructed. The NS1 protein was finally expressed in 293T. Conclusion:The full-length NS1 gene was obtained as well as its recombinant eukaryotic expression plasmid was successfully constructed. The construction of eukaryotic expression plasmid of NS1 gene made it possible to further study the function of NS1 protein and the mechanism of diseases induced by influenza A virus.