Background: To evaluate the role of real-time polymerase chain reaction (PCR) in pleural fluid (PE) specimens as non-in-vasive method for pleural tuberculosis (TB). Methods: We analyzed medical records of patients who underwent thoracentesis to evaluate unilateral PE from Jan, 2009 to Dec, 2010. The patients with TB empyema and positive smear for acid fast bacilli (AFB) in sputum were excluded. Results: In a total of 210 patients who underwent thoracentesis, a PE specimen was submitted for real-time TB-PCR. Overall, 56 (26.7%) patients had pleural TB and 154 had PE secondary to other diseases. TB was confirmed using positive culture for acid-fast bacilli (AFB) (n=7) or favorable clinical response to anti-TB medication (n=49). Of all patients, only two patients had positive real-time TB-PCR. They had negative AFB smear and culture of PE and were finally diagnosed as pleural TB. Therefore, sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of real-time-TB-PCR testing in PE were 3.57%, 100%, 100% and 72.04%, respectively. In addition, three patients have positive AFB smear in PE and were finally confirmed to have pleural TB. Therefore, sensitivity, specificity, PPV and NPV of PE AFB smear were 5.36%, 100%, 100% and 74.4%, which were not different from those of real-time TB-PCR. Conclusions: The sensitivity of real-time TB-PCR of PE was low. However, real-time TB-PCR could be an additional non-invasive assay for fast confirmation of pleural TB, especially when it is added to AFB smear.