The sec2 gene was cloned from the genomic DNA of Staphylococcus aureus by PCR and ligated with the pGEM-T vector and confirmed by DNA sequencing. The expression vector pET-28a-SEC2 was constructed to express rSEC2 protein in E.coli. Puried rSEC2 protein was taken to bioactivity analysis. The cloned sec2 gene contains 717 nucleotides coding 239 amino acids which was accordance with that reported in GenBank. Cloned sec2 sequence was submitted to GenBank and the Accession number is AY450554. Soluble rSEC2 protein could be expressed effectively in BL21(DE3) and puried by Ni-NTA. Western blotting, PBMC proliferation assay and in vitro anti-tumor assay revealed that rSEC2 has the function of native SEC2. It was concluded that the whole sec2 gene was cloned and expressed in E.coli. Puried rSEC2 protein was proved to have the equivalent function of native SEC2.