Objective: The interferon-γ (IFN-γ) release assays still fail to meet the diagnostic needs of etiology negative tuberculosis (TB) patients. The purpose of this paper is to explore the value of the IL-2-enzyme-linked immunospot (ELISPOT) assay in the auxiliary diagnosis of TB. Methods: We recruited 127 TB patients, 49 non-TB patients with other diseases (NTB), and 198 healthy people, whose peripheral blood mononuclear cells were isolated and stimulated by Mtb-specific antigen CFP-10/ESAT-6 fusion protein to study the changes of T cells secreting IFN-γ and Interleukin-2 (IL-2) by ELISPOT method. Results: The combination of IFN-γ and IL-2 had an area under the curve (AUC) of 0.867 (IFN-γ: 0.835, IL-2: 0.866) in the diagnosis of TB and could increase the sensitivity of IFN-γ detection alone from 79.5% to 86.6%. The cut-off value for IL-2 and IFN-γ was 16. IL-2 could increase the positive diagnostic rates of IFN-γ-ELISPOT (-) in the etiologically positive and negative ATB patients by 9.4% and 7.1%. In the ATB group, the positive rates and the numbers of spot-forming cells (SFC) of IFN-γ and IL-2 showed significant differences compared to the normal control subjects and NTB group (P<0.05). And SFCs of IFN-γ-ELISPOT were significantly higher than that of IL-2-ELISPOT in ATB patients. Before the treatment, the SFC values of the etiologically positive TB patients were higher than those of the etiologically negative patients. However, this situation was totally reversed after anti-TB treatment. After effective treatment, the positive rates and SFC values of IFN-γ and IL-2 in TB patients decreased and exhibited a decreasing and increasing trend in etiologically positive and negative TB patients, respectively. Conclusions: The combination of IL-2 and IFN-γ could improve the diagnostic sensitivity of single-factor detection in TB patients and these two cytokines could be used as effective biomarkers for the treatment monitoring in TB patients.