Background: Simple, rapid, and accurate diagnosis of tuberculous pleural effusion (TPE) remains challenging. This study aimed to determine the accuracy of IFN-gamma in diagnosing TPE. Methods: We quantified the expression of interferon-gamma (IFN-gamma) in blood (B), adenosine deaminase (ADA), and IFN-gamma in pleural effusions (PE) from 25 TPE patients and 31 non-TPE patients using a combination of immunological assays and flow cytometric analysis. The diagnostic performance of these three biomarkers was evaluated using receiver operating characteristic (ROC) curves. Results: We found that IFN-gamma levels in blood and pleural fluid were higher in the TPE group than in the non-TPE group. The mean concentration of IFN-gamma in pleural fluid of the TPE group was 3140.90 (1817.94, 6611.05) pg/ mL, while that of the non-TPE group was 4.91 (0.69, 8.6) pg/mL), and the difference was statistically significant (z = 6.39, P < 0.001). The mean blood IFN-gamma was 40.19 (16.45, 59.08) pg/mL in the TPE group and 2.76 (1.96, 6.02) pg/mL in the non-TPE group, which was statistically different (z = 5.12, P < 0.001). The area under the ROC curve (AUC) for pleural fluid IFN-gamma, blood IFN-gamma, and ADA were 0.999 (95 % CI: 0.994-1.00), 0.901 (95 % CI: 0.798-1.00) and 0.996 (95 % CI: 0.987-1.00), respectively. Conclusion: This study confirms that IFN-gamma has high diagnostic validity in patients with TPE and can potentially be an excellent biomarker.