Diagnosing pleural tuberculosis (plTB) might be difficult due to limited sensitivity of conventional microbiology tools. As M. tuberculosis (MTB)-specific T cells are recruited into pleural space in plTB, their detection may provide useful clinical information. To this aim, in addition to standard diagnostic tests, we used the QuantiFERON-TB Gold In-Tube (QFT-IT) test in blood and pleural effusion (PE) samples from 48 patients with clinical suspicion of plTB, 18 (37.5%) of whom had confirmed plTB. Four of them (22.2%) tested positive with a nucleic acid amplification test for MTB. The tuberculin skin test was positive in most confirmed plTB cases (88.9%). Positive QFT-IT tests were significantly more frequent in patients with confirmed plTB, as compared to patients with an alternative diagnosis, both in blood (77.7 vs 36.6%, p=0.006) and in PE samples (83.3% vs 46.6%, p=0.02). In addition, both blood and PE MTB-stimulated IFN-gamma levels were significantly higher in plTB patients (p=0.03 and p=0.0049 vs non-plTB, respectively). In blood samples, QFT-IT had 77.8% sensitivity and 63.3% specificity, resulting in 56.0% positive (PPV) and 82.6% negative (NPV) predictive values. On PE, QFT-IT sensitivity was 83.3% and specificity 53.3% (PPV 51.7% and NPV 84.2%). The optimal AUC-derived cut-off for MTB-stimulated pleural IFN-gamma level was 3.01 IU/mL (77.8% sensitivity, 80% specificity, PPV 68.4% and NPV 82.8%). These data suggest that QFT-IT might have a role in ruling out plTB in clinical practice.