To clone Rv0859 gene of Mycobacterium tuberculosis and purify its fusion protein. Genome DNAs of H37Rv was extracted. Primers for Rv0859 gene was designed, and DNA fragments encoding Rv0859was obtained by PCR and inserted into proeukaryotic expression plasmid pET30a followed by digestion with Hind III and BamH I. pET30a-Rv0859 is connected by T4 ligase which was transformed into Escherichia coli JF1125. Sequence analysis was performed to verify the recombinant plasmid. The recombinant plasmid was transfected into Escherichia coli BL21. After induction with IPTG, the expression of Rv0859 protein was identified by SDS-PAGE and mass spectrum. The expressed protein was the maximum when induced with 0.05 mol/L IPTG for 4 h at 37°C. The polyclonal antibodies were prepared to detect the subcellular localization by Western-blotting. The result is the prokaryotic expression vector pET30a-Rv0859 was constructed successfully and the recombinant Rv0859 was expressed heavily. The result of Western-blotting indicated that the protein located on the cell envelope partly and little on the cell well. The study laid the foundation for application in the tuberculosis.