We established that an MTB-specific triple color FluoroSpot (IFN-γ/IL-2/TNF-α) assay can simultaneously detect three cytokines closely related to TB infection status and found that its diagnostic accuracy was better than that of T-SPOT.TB, which may play an important role in assisting the diagnosis of ATB, especially in differentiating ATB from LTBI. This evidence concerns the gene TNF and tuberculosis.