T-SPOT.TB didn't perform a perfect diagnosis for active tuberculosis (ATB), and some factors may influence the results. We did this study to evaluate possible factors associated with the sensitivity and specificity of T-SPOT.TB, and the diagnostic parameters under varied conditions. Patients with suspected ATB were enrolled prospectively. Influencing factors of the sensitivity and specificity of T-SPOT.TB were evaluated using logistic regression models. Sensitivity, specificity, predictive values (PV), and likelihood ratios (LR) were calculated with consideration of relevant factors. Of the 865 participants, 205 (23.7%) had ATB, including 58 (28.3%) microbiologically confirmed TB and 147 (71.7%) clinically diagnosed TB. 615 (71.7%) were non-TB. 45 (5.2%) cases were clinically indeterminate and excluded from the final analysis. In multivariate analysis, serous effusion was the only independent risk factor related to lower sensitivity (OR = 0.39, 95% CI: 0.18-0.81) among patients with ATB. Among non-TB patients, age, TB history, immunosuppressive agents/glucocorticoid treatment and lymphocyte count were the independent risk factors related to specificity of T-SPOT.TB. Sensitivity, specificity, PV+, PV-, LR+ and LR- of T-SPOT.TB for diagnosis of ATB were 78.5%, 74.1%, 50.3%, 91.2%, 3.0 and 0.3, respectively. This study suggests that influencing factors of sensitivity and specificity of T-SPOT.TB should be considered for interpretation of T-SPOT.TB results.