To obtain purified recombinant Rv3369 protein by means of expressing the Rv3369 protein of Mycobacterium tuberculosis in E. coli. The gene coding Rv3369 protein was amplified by polymerase chain reaction (PCR), then was inserted into an expression vector pET28a to get recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed product was indentified by SDS-PAGE and purified by Ni- NTA His. Bind Resin. The sequence of Rv3369 in recombinant plasmid was the same with GenBank's report. The molecular mass of the product is 19.5kDa, which accounts for about 20% in the thalli proteins, and its purity is more than 90% analyzed by SDS-PAGE and laser scanning. The yield of recombinant protein is 1.56mg from 100mL of culture. Compared with other methods, purity of the recombinant protein is higher through affinity chromatography.