The extremely slow growth rate of Mycobacterium tuberculosis (Mtb) challenges traditional methods for tuberculosis (TB) diagnosis. Here, we assessed the efficacy of a previously developed Mtb antigen-specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) performed on bronchoalveolar lavage fluid (BALF) from a cohort of 414 patients including 333 PTB patients (202/333 were sputum culture positive) for the diagnosis of PTB. We could confirm that antigen-specific IFN-γ-producing CD4⁺ T cells were concentrated significantly in BALF mononuclear cells (BALMC) compared with that in peripheral blood mononuclear cells (PBMC) assayed in parallel, but not those of CD8⁺ T cells both in sputum culture-negative and positive PTB. The magnitude of IFN-γ responses in the BALF was associated with bacterial load, and 9/202 of PTB with endobronchial TB (EBTB) were slightly reduced by the anti-TB treatment. Moreover, antigen-specific IFN-γ ELISPOT performed on BALMC showed higher sensitivity than PBMC ELISPOT. In addition, the differences of the BALMC ELISPOT between PTB and PTB with diabetes were not found, whereas PBMC IFN-γ responses were decreased in PTB with diabetes. Combined with the microbiological detection in BALF, such as microscopy and culture, the BALMC ELISPOT offers the opportunity for the more accurate diagnosis of PTB, especially those with clinical comorbidities.