Tuberculosis (TB) is one of most important chronic infectious diseases caused by Mycobacterium tuberculosis and remains a major global health problem. In the study, we developed the DNA vaccine encoding fusion protein of antigen 85 A and 6 kDa early secretory antigen target of M. tuberculosis as well as the cytokine IL-21 to investigate its immune protective efficacy against M. tuberculosis challenge in mice after the DNA vaccine priming and Bacille Calmette-Guérin (BCG) boosting. Compared with the different control groups, the intranasal DNA vaccine priming twice and BCG boosting once markedly increased the cytotoxicities of natural killer cells and splenocytes and enhanced the interferon-γ level in the splenocyte supernatant as well as sIgA level in bronchoalveolar lavage in the vaccinated mice. Importantly, this heterologous prime-boost strategy significantly decreased the bacterial load in the mouse lungs in contrast to that of intranasal or subcutaneous BCG immunization alone. These findings provide further approaches for mucosal-targeted prime-boost vaccination to fight against TB.