The study investigated the diagnostic value of Mycobacterium tuberculosis MPT64 antigen in bronchoalveolar lavage (BALF) in 176 patients, 104 with pulmonary tuberculosis (PTB) and 72 without tuberculosis as controls. The PTB group consisted of 59 bacteriologically confirmed cases (BC-PTB) and 45 clinically diagnosed with negative pathogens (CD-PTB). MPT64 concentration was detected by enzyme-linked immunosorbent assay (ELISA) and optimal limits were determined using ROC curves. MPT64 showed a sensitivity of 59.62% (95% CI: 50.01–68.54%), higher than AFB microscopy (26.92%, P < 0.001) and comparable to Xpert MTB/RIF (56.73%, P = 0.673), specificity 88.89% (95% CI: 79.58–94.26%). lower than AFB and Xpert (100%, P = 0.006). The sensitivity for BC-PTB was 64.41% (95% CI: 51.66–75.40%) and for CD-PTB 53.33% (95% CI: 39.08–67.06%), P = 0.314. Xpert MTB/RIF Ct values and MPT64 concentration show a negative correlation (R = -0.719, P < 0.01), as well as AFB and MPT64 a positive correlation (R = 0.777, P < 0.0001). The MPT64 antigen in BALF has potential in the diagnosis of pulmonary tuberculosis, especially in bacteriologically negative cases.