Increasing evidences in animals and humans suggest that CD8⁺ T cells contribute significantly to immune defenses against Mycobacterium tuberculosis (Mtb). In the present study, we found that without any stimulation, CD8⁺ T cells in pleural fluid cells (PFCs) expressed significantly higher levels of CD69 than PBMCs from patients with tuberculous pleurisy (TBP). CD8⁺CD69⁺ T cells expressed significantly higher levels of CD45RO and HLA-DR and lower levels of CD45RA than CD8⁺CD69⁻ T cells, demonstrating that CD8⁺CD69⁺ T cells were activated memory cells. Furthermore, we found higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8⁺CD69⁺ T cells compared with CD8⁺CD69⁻ T cells, suggesting that the expression of CCR6 and reduced expression of CCR7 and CD62L might facilitate the migration of circulating CD8⁺CD69⁺ T cells into tuberculous pleural space. Importantly, following stimulation with culture filtrate protein of 10 kDa (CFP10) peptides, CD8⁺CD69⁺ T cells but not CD8⁺CD69⁻ T cells expressed CD107a/b, IFN-γ and TNF-α, demonstrating that CD8⁺CD69⁺ T cells were MTB-specific cells. In addition, the majority of CD8⁺CD69⁺ T cells were dominated by polyfunctional T cells. In summary, we demonstrated that CD69 as a useful marker for MTB-specific CD8⁺ T cells in PFCs from patients with TBP enabled a direct ex vivo estimation of the quantity, as well as the quality, of MTB-specific CD8⁺ responses.