Objective：To construct a recombinant multiepitope fusion polypeptide containing 6 HLA-A*0201 restricted CTL epitopes derived from Mycobacterium tuberculosis(Mtb.) antigens and express it in E.coli. Methods: The full-length sequence encoding 6 HLA-A*0201 restricted CTL epitopes were derived from Mtb. antigens Rv0309, Rv0173, and Ag85A, Ag85C; pan DR Th epitope(PADRE), Trojan peptide, and furin-sensitive linker RVKR were synthesized, amplified by polymerase chain reaction(PCR), inserted into the expression vector pGEX-4T-1, and transformed into E.coli BL21(DE3). After induced by isopropylthio-β-D-galactoside(IPTG), the recombinant protein expression was examined by SDS-PAGE and Western blot. Results: The recombinant multiepitope fusion polypeptide plasmid was successfully constructed and expressed(about 40 000) in E.coli BL21(DE3). Conclusion: The recombinant multiepitope fusion polypeptide may provide a basis for developing novel TB vaccine.