Objective：To modify the monoantigen DNA vaccine of Mycobacterium tuberculosis with ubiqutin,so as to obtain more potent immune response. Methods: We constructed Mycobacterium tuberculosis MPT64 antigen DNA vaccine (pM) and ubiquitinMPT64 fusion gene DNA vaccine (pUM).The constructed DNA vaccines were intramuscularly inoculated into female BALB/c mice separately. The serum antibodies(including IgG,IgG1,and IgG2a),cytokines (IFNγ,IL4) and cytotoxic T lymphocyte(CTL) response were determined in immunized mice. Results: The IgG level in the pM group was higher than that in the pUM group（P<0.01） and the ratio of IgG2a/IgG1 in the pM group was lower than that in the pUM group（\[2.16±0.3\] vs \[4.48±0.4\],P<0.05）.The IFNγ level was higher (P<0.01） and the IL4 level was lower (P<0.01） in the pUM group than those in the pM group. Furthermore, the pUM group had higher CTL activity than the pM group. It was indicated that the fusion gene DNA vaccine induced weaker humoral immune response but stronger cellular immune response compared to single gene DNA vaccine. Conclusion: The fusion gene DNA vaccine constructed in the present study might be more effective for prevention against tuberculosis than the single gene DNA vaccine.