Objective To investigate the values of mycobacterium tuberculosis/rifampicin resistance gene(Xpert MTB/RIF) detection, T cell spot test ( T-SPOT.TB) and adenosine deaminase (ADA) assay in detection of pleural fluid in the diagnosis of pleural tuberculosis. Methods A total of 167 patients with pleural fluid admitted to Department of Infective Diseases of Beijing Geriatric Hospital from January 2009 to April 2021 were recruited in this study, and 114 of them were diagnosed with tuberculous pleurisy (TBP group) and 53 cases were not (non-TBP group). Every patient was tested with Xpert MTB/RIF detection, T-SPOT.TB and ADA assays in pleural effusion. SPSS statistics 23.0 was used to perform the statistical analysis. Student′s t test, rank-sum test or Chi-square test was employed for intergroup comparison based on different data types. Results The highest sensitivity was observed in ADA assay (73.68%), followed by Xpert MTB/RIF assay (37.72%) and then Roche culture (23.68%), with statistical differences among them (χ²=29.880,5.277; P<0.05). There was no significant difference in the sensitivity between T-SPOT.TB (81.58%) and ADA assays (73.68%,χ²=2.046; P＞0.05).The specificity was significantly higher in Xpert MTB/RIF assay (100.00%) and Roche culture (100.00%) than T-SPOT.TB assay (81.13%, χ²=11.042,11.042; P＜0.05),and also higher than that of ADA assay (71.70%, χ²=17.473,17.473; P＜0.05). But there was no significant difference in the specificity between T-SPOT .TB (81.13%) and ADA assays (71.70%, χ²=1.309; P＞0.05). When the three methods were connected in series, the detection sensitivity of T-SPOT.TB and ADA assays was obviously higher than that of Xpert MTB/RIF and ADA assays (60.53% vs 35.96%, χ²=20.081, P＜0.001). In parallel connection, the sensitivity of T-SPOT.TB or ADA assays (94.74%) was notably higher than that of ADA assay alone (73.68%) or T-SPOT.TB assay alone (81.58%, χ²=19.000,9.453; P<0.05). No statistical differences were seen in the sensitivity of T-SPOT.TB or ADA or Xpert MTB/RIF (96.49%) with sensitivity of T-SPOT.TB or ADA (94.74%,χ²=0.418, P＞0.05). There was no significant difference in specificity between the two or three tests in series (P＞0.05).In parallel connection, T-SPOT.TB or ADA(62.26%) and T-SPOT TB or ADA or Xpert MTB/RIF had the same specificity (62.26%), which were lower than that of Xpert MTB/RIF or T-SPOT TB (81.13%), and the difference was statistically significant(χ²=4.649,4.649; P＜0.05); and there was no significant difference in other groups in pairwise comparison of specificity (P＞0.05). Conclusion T-SPOT.TB and ADA assays for pleural fluid patients obtain higher sensitivity and specificity in the diagnosis of tuberculous pleurisy, while Xpert MTB/RIF has lower diagnostic value. Combined detection of pleural fluid with T-SPOT.TB and ADA assay can improve the sensitivity and better reduce the missed diagnosis rate of tuberculous pleurisy.