Tuberculosis (TB) is the number one infectious disease killer worldwide. Bacillus Calmette-Guérin (BCG) is the only vaccine currently used in clinical practice to prevent TB, but it still has limitations in preventing latent infection and reactivation of TB. The investigation focused on the selection of the Rv0081 antigen linked to latency, leading to the successful creation of the prokaryotic expression recombinant plasmid pET30b-Rv0081 for subsequent expression and purification processes. Antigen specificity was additionally validated through the implementation of whole blood IFN-γ release assay (WBIA), enzyme-linked immunosorbent assay (ELISA), and flow cytometry analysis. rRv0081 induced individuals infected with Mycobacterium tuberculosis (M. tb) to exhibit markedly elevated levels of IFN-γ in their peripheral blood compared to the control group of healthy individuals. Mice were co-immunized with rRv0081 and DMT, and serum specific antibodies, levels of cytokines secreted by splenocytes and the number of multifunctional T cells in splenocytes were detected. The study findings indicated a notable elevation in the secretion levels of IFN-γ, TNF-α, and IL-2 cytokines from splenocytes, along with increased levels of IgG and its subclasses in the serum of mice belonging to the BCG + rRv0081/DMT group compared to its control group. Furthermore, there was a significant increase in the count of single-positive and double-positive CD4+ and CD8+ T cells stimulated to generate IFN-γ and TNF-α in the BCG + rRv0081/DMT group when contrasted with the BCG group. This suggests that rRv0081/DMT could be a potential subunit vaccine candidate that may serve as an effective booster vaccine after primary immunization against BCG.