Objective To evaluate the value of T lymphocytes detection by flow cytometry in the diagnosis of non-tuberculosis mycobacteria (NTM) disease.Methods Totally, 162 cases of NTM pulmonary disease (NTM group), 143 cases of bacterial positive pulmonary tuberculosis (PTB group), 146 cases of tuberculous pleurisy (tuberculous pleurisy group),81 cases of tuberculous meningitis (tuberculous meningitis group) and 175 cases of other pulmonary diseases (control group, including 101 patients with bacterial pneumonia, 58 patients with mycoplasma pneumonia, 11 patients with bronchiectasis and 5 patients with pneumoconiosis) were retrospectively collected in Xi’an Chest Hospital from January 2013 to March 2019. The results of T lymphocytes detected by flow cytometry in 5 groups were summarized and comparatively analyzed. SPSS 23.0 software was used to statistically analysis, and the measurement data of non-normal distribution were expressed by median (quartile) (M(Q1,Q3)). Kruskal-Waills H test was used to compare the differences of T lymphocyte flow cytometry results between 5 groups as well as between the NTM group and other groups. P<0.05 was considered statistically significant.Results In the NTM group, PTB group, tuberculous pleurisy group, tuberculous meningitis group and control group, the total T lymphocytes were 1276.00 (987.00,1563.00),907.00 (661.00,1231.25),793.00 (556.50,1080.75),574.00 (296.00,883.00),and 1904.00 (1611.50,2093.50)cells/μl; CD4 + T lymphocyte counts were 868.00 (599.00,997.00),546.00 (384.75,744.00),445.00 (301.75,645.00),294.00 (134.00,462.00),and 1497.00 (925.50,1757.50) cells/μl;CD8 + T lymphocyte counts were 369.00 (252.00,573.00), 289.50 (192.00,400.75), 303.50 (206.75,441.00),243.00 (119.00,427.00), and 417.00 (279.50,644.50) cells/μl; and CD4 +CD8 + T lymphocytes counts were 11.00 (7.00, 18.00), 6.00 (4.00, 11.00), 8.00 (6.00, 13.00), 5.00 (2.00, 9.00), and 9.00 (6.00, 12.50) cells/μl. The data of each group were the highest in the control group, followed by the NTM group, and the lowest in each tuberculosis groups. There were significant differences in total T lymphocytes, CD4 + and CD8 + T lymphocytes among the five groups as well as between NTM group and PTB group, tuberculous pleurisy group, tuberculous meningitis group and control group (H=339.898, 5.291, 6.968, 8.323, -7.662, Ps=0.000; H=384.960, 5.973, 8.777, 10.128, -6.641, Ps=0.000; H=66.345, 4.632, 3.503, 5.530, -0.897, Ps=0.000, respectively). There were significant differences in CD4 +/CD8 + among the five groups as well as between NTM group and PTB group, tuberculous pleurisy group, and tuberculous meningitis group (H=59.508, 5.453, 3.189, 6.824, Ps<0.05). Conclusion The number of T lymphocytes in patients with NTM disease is lower than that in patients with other pulmonary diseases (control group), and higher than that in patients with tuberculosis in three groups. It can be used to judge the immune status of patients and has a certain auxiliary diagnostic value.