Research dedicated to diagnostic reagents and vaccine development for tuberculosis (TB) is challenging due to the paucity of immunodominant antigens that can predict disease risk and exhibit protective potential. Therefore, it is crucial to identify T-cell epitope-based Mycobacterium tuberculosis (MTB) antigens characterized by specific and prominent recognition by the immune system. In this study, we constructed a T-cell epitope-rich tripeptide-splicing fragment (nucleotide positions 131-194, 334-377, and 579-643) of Rv2201 (also known as the 72 kDa AsnB)from the MTB genome, ultimately yielding the recombinant protein Rv2201-519 in Escherichia coli BL21 (DE3). Subsequently, we gauged the recombinant protein's ability to detect tuberculosis infection through ELISpot and assessed its immunostimulatory effect on mouse models using flow cytometry and ELISA. Our results indicated that Rv2201-519 possessed promising sensitivity; however, the sensitivity was lower than that of a commercial diagnostic kit containing ESAT-6, CFP-10, and Rv3615c (80.56 % vs. 94.44 %). The Rv2201-519 group exhibited a propensity for a CD4 Th1 cell immune response in inoculated BALB/c mice that manifested as higher levels of antigen-specific IgG production (IgG2a/IgG1 > 1). In comparison to Ag85B, Rv2201-519 induced a more robust Th1-type cellular immune response as evidenced by a notable rise in the ratio of IFN-γ/IL-4 and IL-12 cytokine production and increased CD4 T cell activation with a higher percentage of CD4IFN-γ T cells. Rv2201-519 also induced a higher level of IL-6 compared with Ag85B, a higher percentage of CD8 T cells specific for Rv2201-519, and a lower percentage of CD8IL-4 T cells. Collectively, the current evidence suggests that Rv2201-519 could potentially serve as an immunodominant protein for tuberculosis infection screening, laying the groundwork for further evaluation in recombinant Bacillus Calmette-Guérin (BCG) and subunit vaccines against MTB challenges in future studies.