A specific immunoassay method with the colloidal gold labeling technique has been developed more and more for tuberculosis (TB) diagnosis. The aim of this multicenter clinical evaluation was to evaluate the performance of a new serological diagnostic kit (the Trustline TB IgG/IgM Rapid Test kit) for the detection of Mycobacterium tuberculosis infection in China, with the Aupu TB Ab (IgG) Colloidal Gold Test kit being used as a control. A total of 1,009 specimens were collected from three TB hospitals, including 628 patients with TB, and 219 non-TB and 162 healthy patients as negative controls. According to the clinical diagnostic results, the sensitivities of the Trustline kit and the Aupu kit were 61.3 % and 53.7 %, respectively. Using the bacteriological test results as the "gold standard" to compare the results of the two kits, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden index (YI) were 77.60 %, 79.8 %, 73.31 %, 81.50 %, and 0.574 by the Trustline kit, and 67.86 %, 88.9 %, 83.27 %, 77.40 %, and 0.568 by the Aupu kit, respectively. Further, the sensitivity of the Trustline kit and the Aupu kit for the smear staining and the bacterial culture being positive was 75.6 %, 76.6 % and 65.6 %, 66.5 %, and for the negative result, it was 53.8 %, 50.9 % and 47.5 %, 45.0 %, respectively. Additionally, 35 specimens were IgM-positive by the Trustline kit; of these, 30 (4.8 %) were from patients with TB and 5 (1.3 %) were from individuals without TB. The results showed that the experimental test had a much higher sensitivity than the other commercial test and exhibited a good detection rate for M. tuberculosis infection. Therefore, this kit can be used in the supplementary diagnosis and screening of TB.