Tuberculosis (TB) remains a leading global cause of death from infectious disease. Current enzyme-linked immunospot assay (ELISPOT)-based interferon-gamma release assays (IGRAs) rely on pooled synthetic peptides, which pose challenges for large-scale good manufacturing practice (GMP) production and quality control. To address these limitations, we developed recombinant overlapping peptide (ROP) technology and evaluated its diagnostic performance in a multi-center clinical trial in China (September 2021-August 2022). Two reagents, representing distinct antigen presentation pathways, demonstrated high concordance. ROP-TB showed excellent agreement with T-SPOT.TB, yielding positive, negative, and overall agreement rates of 95.29%, 93.51%, and 94.49%, respectively (κ = 0.889, p < 0.001). The sensitivity and specificity of ROP-TB were 88.03% and 76.79%. Notably, ROP-TB elicited a significantly stronger immune response amplitude in individuals with presumed latent and drug-resistant TB. Our findings suggest that ROP-TB is a viable auxiliary diagnostic and screening tool, offering performance comparable to T-SPOT.TB with enhanced detection of latent and resistant strains.