Interferon-gamma release assays (IGRAs) have proven to be useful to accurately detect Mycobacterium tuberculosis (Mtb) infection, but they cannot reliably discriminate between active tuberculosis (TB) and latent tuberculosis infection (LTBI). This study aims to test whether Mtb-specific tumor necrosis factor-alpha (TNF-alpha) could be used as a new tool for the rapid diagnosis of active TB disease. The secretion of TNF-alpha by Mtb-specific antigen-stimulated peripheral blood mononuclear cells (PBMCs) of sixty seven participants was investigated in the study. Our results showed that the total measurement of TNF-alpha secretion by Mtb-specific antigen-stimulated PBMCs is not a good biomarker for active TB diagnosis. However, we found that calculation of Mtb-specific TNF-alpha not only distinguish between active and latent TB infection, but also can differentiate active TB from non-TB patients. Using the cutoff value of 136.9 pg/ml for Mtb-specific TNF-alpha, we were able to differentiate active TB from LTBI. Sensitivity and specificity were 72% and 90.91%. These data suggest that Mtb-specific TNF-alpha could be a potential biomarker for the diagnosis of active TB disease.