The performance of T-SPOT.TB (T-SPOT) assay in diagnosing pleural tuberculosis (plTB) is inconsistent. In this study, we compared the performance of peripheral blood (PB) and pleural fluid (PF) T-SPOT assay in diagnosing plTB. Between July 2017 and March 2018, 218 and 210 suspected plTB patients were prospectively enrolled from Wuhan (training) and Guangzhou (validation) cohort, respectively. PB T-SPOT, PF T-SPOT, and other conventional tests were simultaneously performed. Our data showed the performance of PB T-SPOT in diagnosing plTB was limited, especially with low sensitivity. However, the results of early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in PF T-SPOT were significantly increased compared with those in PB T-SPOT in plTB patients. If using 76 as the cutoff value of MAX (the larger of ESAT-6 and CFP-10) in Wuhan cohort, the sensitivity and specificity of PF T-SPOT to diagnose plTB were 89.76 and 96.70%, respectively. The diagnostic accuracy of PF T-SPOT was better than other routine tests such as pathogen detection methods and biochemical markers. The diagnostic accuracy of PF T-SPOT in Guangzhou cohort was similar to that in Wuhan cohort, with a sensitivity and specificity of 91.07 and 94.90%, respectively. Furthermore, CD4(+) T cells were more activated in PF compared with PB, and the frequency of mycobacterium tuberculosis-specific CD4(+) T cells in PF was significantly higher than that in PB in plTB patients. In conclusion, the performance of PF T-SPOT is obviously better than PB T-SPOT or other laboratory tests, which suggests that PF T-SPOT assay has been of great value in the diagnosis of pleural tuberculosis.