DNA vaccines have been extensively studied as preventative and therapeutic interventions for various infectious diseases such as tuberculosis, HIV/AIDS and influenza. Despite promising progresses made, improving the immunogenicity of DNA vaccine remains a technical challenge for clinical development. In this study, we investigated a tuberculosis DNA vaccine BER^opt, which contained a codon-optimized fusion immunogen Ag85B-ESAT-6-Rv2660c for enhanced mammalian cell expression and immunogenicity. BER^opt immunization through in vivo electroporation in BALB/c mice induced surprisingly high frequencies of Ag85B tetramer⁺ CD8⁺ T cells in peripheral blood and IFN-γ-secreting CD8⁺ T cells in splenocytes. Meanwhile, the BER^opt vaccine-induced long-lasting T cell immunity protected BALB/c mice from high dose viral challenge using a modified vaccinia virus Tiantan strain expressing mature Ag85B protein (MVTT-m85B) and the virulent M. tb H37Rv aerosol challenge. Since the BER^opt DNA vaccine does not induce anti-vector immunity, the strong immunogenicity and protective efficacy of this novel DNA vaccine warrant its future development for M. tb prevention and immunotherapy to alleviate the global TB burden.