Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, the Bacille Calmette-Guérin (BCG), is urgently needed. BCG has proven to be an effective recombinant delivery vehicle for foreign antigens because of its ability to induce long-lived specific humoral and cellular immunity. Experimental evidences have revealed that Ag85B, ESAT-6 and Rv2608 are important immunodominant antigens of Mycobacterium tuberculosis and are all promising vaccine candidate molecules. In this study, we have constructed a novel recombinant BCG (rBCG) expressing fusion protein Ag85B-ESAT6-Rv2608 and evaluated the immunogenicity of rBCG in C57BL/6 mice. Results show there is strong TB-specific CD4⁺ and CD8⁺ T lymphocytes proliferative response in mice immunized with rBCG vaccine, especially the cytotoxic CD8⁺ T cells playing an important role in protection against TB. And rBCG immunization has induced a significantly strong Th1 immune response, characterized by the increased ratio of IgG2b/IgG1. Results also show that rBCG immunization could increase the secretion of Th1 cytokines such as TNF-α and IL-2 and could decrease the secretion of Th2 cytokine IL-10. Moreover, it was shown that rBCG immunization induced a strong humoral response in mice, characterized by the elevated IgG titre. Therefore, we conclude that this rBCG immunization could increase both cellular immune response and antigen-specific humoral response significantly as compared to BCG immunization in mice. The above results illustrated that rBCG::Ag85B-ESAT6-Rv2608 is a potential candidate against M. tuberculosis for further study.