Tuberculosis (TB) today remains the leading cause of global deaths due to infectious bacterial pathogens. The Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine clinically used to prevent TB. However, its limitations in preventing latent infection and TB reactivation mean that it does not provide comprehensive protection. In this study, we successfully constructed and expressed the multistage fusion protein, SHR3, and used whole blood IFN-γ release assay (WBIA) with flow cytometry to detect antigen specificity, further confirmed by enzyme-linked immunosorbent assay (ELISA). SHR3 and its subfractional proteins stimulated the level of IFN-γ production by lymphocytes from M. tb-infected patients, inducing the production of single-positive and double-positive CD4⁺ and CD8⁺ T cells with IFN-γ and IL-2, at levels significantly higher than those of healthy controls. The fusion protein and complex adjuvant group (SHR3/DMT) induced mice to produce significantly higher levels of IgG antibodies and their subclasses, with IgG2a/IgG1 results showing a convergent Th1-type response; mice in the BCG + SHR3/DMT group induced secretion of the highest levels of IL-2, and TNF-α, irrespective of stimulation with purified protein derivative or SHR3. These findings suggest that SHR3/DMT could be a potential subunit vaccine candidate that may serve as an effective booster vaccine after BCG primary immunization.