Objective: To detect the expression of T lymphocyte subsets and T cell surface co-signaling molecules in peripheral blood of populations with different Mycobacterium tuberculosis infection statuses and analyze their clinical significance. Methods: A prospective study design was employed. A total of 22 patients with pulmonary tuberculosis (TB group), 20 individuals with latent Mycobacterium tuberculosis infection (LTBI group), who met the inclusion and exclusion criteria, were enrolled at Beijing Chest Hospital Affiliated to Capital Medical University between August 3, 2023 and October 30, 2024. In addition, 22 healthy volunteers (HC group) who underwent physical examination during the same period were recruited as controls. Peripheral blood mononuclear cells from the three groups were analyzed using full-spectrum multiparametric flow cytometry to investigate the proportions of T lymphocytes and the distributions of 13 co-signaling molecules on T cells surface among different populations. Results: The levels of CD3+ T cells in the TB group ((53.23±16.15) %) and CD4+ T cells ((33.06±9.58) %) were significantly lower than those in the HC group ((65.59±13.94) % and (40.39±11.01) % respectively), and the level of CD8+ T cells ((16.88±7.59) %) was lower than that in the HC group ((24.07±8.86) %) and the LTBI group ((24.56±7.09) %), the differences were statistically significant (q=4.156, P=0.013; q=3.594, P=0.036; q=4.267, P=0.010; q=4.450, P=0.007). In CD4+ T cells, the expression level of CTLA-4 in the TB group (0.42% (0.21%, 1.92%)) was significantly higher than that in the HC group (0.20% (0.14%, 0.34%)) and the LTBI group (0.14% (0.07%, 0.20%)), the expression level of CD160 in the TB group (0.00% (0.01%, 0.05%)) was significantly lower than that in the HC group (0.08% (0.01%, 0.17%), the proportion of TIGIT in the LTBI group ((20.87±6.11) %) was significantly higher than that in the HC group ((13.93±5.28) %) and the TB group ((12.92±5.13) %), and the differences were all statistically significant (Z=2.503, P=0.037; Z=4.548, P<0.001; Z=2.447, P=0.043; q=5.877, P<0.001; q=4.903, P=0.003). In CD8+ T cells, the expression level of CTLA-4 in the TB group (1.03% (0.22%, 1.63%)) was significantly higher than that in the HC group (0.10% (0.04%, 0.46%)) and the LTBI group (0.08% (0.05%, 0.12%)), and the differences were statistically significant (Z=3.477, P=0.002; Z=4.279, P<0.001). Among the co-stimulatory signaling molecules, the expression levels of GITR of CD4+ T cells in the TB group and the LTBI group (2.34% (1.36%, 5.20%) and 2.01% (1.64%, 3.80%) respectively) were significantly higher than that in the HC group (1.48% (0.88%, 1.64%)), and the proportions of CD40L of CD4+ T cells, OX40 and ICOS of CD8+ T cells in the TB group (0.07% (0.03%, 0.10%), 0.16% (0.05%, 0.46%) and 7.28% (4.46%, 16.56%) respectively) were significantly higher than those in the HC group (0.02% (0.01%, 0.05%), 0.03% (0.01%, 0.09%), and 2.97% (1.48%, 5.54%) respectively), and the differences were all statistically significant (Z=2.834, P=0.014; Z=3.027, P=0.007; Z=2.825, P=0.014; Z=3.655, P<0.001; Z=3.364, P=0.002). Conclusion: The proportion of T lymphocytes in the peripheral blood of patients infected with Mycobacterium tuberculosis, especially those with active pulmonary tuberculosis, is imbalanced. At the same time, the expression of co-signaling molecules on the surface of T cells also changes, suggesting that such patients have immunosuppression or may have T cell exhaustion. This may provide new directions for development of tuberculosis diagnosis, host-directed immunotherapy, and new tuberculosis vaccine.