To understand the characteristics of the ns1 gene of Avian influenza A virus H5N1 and to prepare NS1 protein in scale, viruses were cultivated in embryonated hen eggs and virion RNA was extracted from allantoic fluid. The complete ns gene was amplified by RT-PCR and analyzed. Sequence analysis demonstrated that the NS1 cDNA contained one open reading frame of 678 bp, which encoded a polypeptide of 225 amino acids. The ns1 gene of Qa/ST/852/01 (H5N1) isolate was closer to that of some H5N1 strains prevalent in southern China in recent years, the homologies of nucleotide and amino acid sequences were 99.0% apprx97.5% and 99.1% apprx97.8%, respectively. Subsequently, ns1 cDNA was obtained by PCR and cloned into pGEX-4T-3 vector to construct recombinant plasmid pGEX-4T-3/NS1 cDNA, and expressed in BL21(DE3) E.coli strain. SDS-PAGE and scan analysis showed recombinant fusion protein GST-NS1 was expressed in soluble form, and fusion protein amounted to 28.5% of the total soluble bacterial protein. The protein was purified by affinity chromatography and the purity was greater than 96%. Western blot analysis of recombinant fusion protein confirmed it could be specifically recognized by antibody of GST. Construction of the prokaryotic expression vector provided a foundation for further studying the function of NS1 protein and preparation of NS1 antibody.