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Mycobacterium tuberculosis infection accounted for 1.3 million deaths worldwide in 2022. Bacillus Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB); however, it has limited protective efficacy in adults. In this study, we constructed a recombinant vaccinia virus expressing Ag85B from M. tuberculosis using a novel attenuated vaccinia virus (KVAC103). We then analyzed the immunogenicity of prime-boost inoculation strategies using recombinant KVAC103 expressing Ag85B (rKVAC85B) compared to BCG. In both rKVAC85B prime-boost and BCG prime-rKVAC85B boost inoculation regimens, rKVAC85B induced the generation of specific immunoglobulin G (IgG) and secretion of interferon-γ by immune cells. In vitro analysis of Mycobacterium growth inhibition revealed a comparable immune-mediated pattern of outcomes. Furthermore, bacterial loads in the lungs were significantly lower in mice inoculated with the BCG prime-rKVAC85B boost than in the BCG-only group following a rechallenge infection with both H37Rv and HN878 strains of M. tuberculosis. These findings collectively suggest that KVAC103, incorporated into a viral vector, is a promising candidate for the development of a novel TB vaccine platform that is effective against multiple M. tuberculosis strains, including H37Rv and HN878, and that rKVAC85B effectively stimulates immune responses against M. tuberculosis infection.