BACKGROUND: Interferon-gamma release assay (IGRA) has been used in latent tuberculosis (TB) infection and TB diagnosis, but the results from different high TB-endemic countries are different. The aim of this study was to investigate the value of IGRA in the diagnosis of active pulmonary TB (PTB) in China. METHODS: We conducted a large-scale retrospective multicenter investigation to further evaluate the role of IGRA in the diagnosis of active PTB in high TB-epidemic populations and the factors affecting the performance of the assay. All patients who underwent valid T-SPOT.TB assays from December 2012 to November 2015 in six large-scale specialized TB hospitals in China and met the study criteria were retrospectively evaluated. Patients were divided into three groups: Group 1, sputum culture-positive PTB patients, confirmed by positive Mycobacterium tuberculosis sputum culture; Group 2, sputum culture-negative PTB patients; and Group 3, non-TB respiratory diseases. The medical records of all patients were collected. Chi-square tests and Fisher's exact test were used to compare categorical data. Multivariable logistic analyses were performed to evaluate the relationship between the results of T-SPOT in TB patients and other factors. RESULTS: A total of 3082 patients for whom complete information was available were included in the investigation, including 905 sputum culture-positive PTB cases, 914 sputum culture-negative PTB cases, and 1263 non-TB respiratory disease cases. The positive rate of T-SPOT.TB was 93.3% in the culture-positive PTB group and 86.1% in the culture-negative PTB group. In the non-PTB group, the positive rate of T-SPOT.TB was 43.6%. The positive rate of T-SPOT.TB in the culture-positive PTB group was significantly higher than that in the culture-negative PTB group (χ2 = 25.118, P < 0.01), which in turn was significantly higher than that in the non-TB group (χ2 = 566.116, P < 0.01). The overall results were as follows: sensitivity, 89.7%; specificity, 56.37%; positive predictive value, 74.75%; negative predictive value, 79.11%; and accuracy, 76.02%. CONCLUSIONS: High false-positive rates of T-SPOT.TB assays in the non-TB group limit the usefulness as a single test to diagnose active TB in China. We highly recommend that IGRAs not be used for the diagnosis of active TB in high-burden TB settings.