The prompt diagnosis of active tuberculosis (ATB) is still a challenge in clinical practice, especially in TB-endemic countries. We prospectively enrolled consecutive patients with suspected pulmonary TB from two tertiary hospitals. Acid-fast staining (AFS), Xpert MTB/RIF (Xpert), Mycobacterium tuberculosis culture, and T-SPOT.TB were simultaneously performed. 226 ATB and 348 non-TB patients were diagnosed in Tongji hospital (test cohort), and 86 ATB and 110 non-TB patients were diagnosed in Guangzhou Chest Hospital (validation cohort). Using ATB as patient group and non-TB as control group, for diagnosis of ATB in Tongji Hospital, the sensitivity of AFS was 17.70% (95% CI: 13.08-23.44%). The sensitivity of Xpert and culture were 53.54% (95% CI: 46.81-60.14%) and 46.46% (95% CI: 39.86-53.19%), respectively. The sensitivity of T-SPOT.TB was 81.42% (95% CI: 75.60-86.14%), but the specificity was 71.55% (95% CI: 66.60-76.04%). Calculation of the ratio of TB-specific antigen to phytohaemagglutinin (TBAg/PHA) of T-SPOT.TB assay increased the specificity but with a loss of sensitivity. Combination of Xpert and culture slightly increased the sensitivity compared to using these methods separately. Combination of Xpert and TBAg/PHA ratio (defined as Xpert positive or TBAg/PHA >= 0.2) increased diagnostic accuracy, and the sensitivity and specificity of combination of them were 85.84% (95% CI: 80.45-89.98%) and 95.98% (95% CI: 93.36-97.59%), respectively. The diagnostic model was also established based on combination of Xpert and TBAg/PHA ratio. The area under the curve of the diagnostic model was 0.952 (95% CI: 0.932-0.973) for diagnosis of ATB, with a sensitivity of 88.05% (95% CI: 83.10-91.98%) and a specificity of 96.26% (95% CI: 93.70-98.00%) when a cutoff value of 0.44 was used in Wuhan cohort. The performance of combination of Xpert and TBAg/PHA ratio was similar in Guangzhou Chest Hospital. Our data suggest that combination of Xpert and TBAg/PHA ratio may be a good algorithm for prompt diagnosis of ATB in high endemic areas.