In order to evaluate the immunogenicity and protective efficacy of tetravalent combination M.tuberculosis DNA vaccine, DNA vaccines encoding Ag85B, MPT64, MPT70 and PstS-3 protein were constructed with eukaryotic expression vector pJW4303. Combination DNA vaccines were inoculated intramuscularly into C57BL/6 mice three times at 3 weeks interval. After 21 days of the third injection, the specific antibody titers against the four antigens were 1∶6 400, 1∶51 200, 1∶6 400 and 1∶6 400. Meanwhile the mixed spleen cells could produce the high antigen-specific IFN-γ level in response to the four antigen proteins, IFN-γ level of Ag85B, MPT64, MPT70 and PstS-3 reached 10 582.14 ng/L, 13 635.97 ng/L, 14 213.15 ng/L and 9 657.35 ng/L respectively. After the last injection, mice were challenged with M.tuberculosis H37Rv. When compared to negative group, the bacterial counts of lung and spleen from the mice vaccinated with tetravalent combination vaccine were reduced about 650 and 130 folds. Microphotographs showed clearly that lungs of mice vaccinated with combination vaccine were much better protected against Mycobacterium tuberculosis challenge than negative mice. The results showed that tetravalent combination M.tuberculosis DNA vaccine elicited both T-cell and humoral immune response, and protected against tuberculosis effectively.