Objective To evaluate the application of a newly approved serological diagnostic kit for tuberculosis (TB) diagnosis:Trustline TB IgG/IgM rapid test kit. Methods In this multiple center study, 1009 serum specimens were collected from three hospitals and tested with Trustline TB IgG/IgM rapid test kit and a commercial TB Ab (IgG) colloid gold test kit (control test). Among the 1009 specimens, 628 were from TB patients and 381 were from non TB individuals. By gold standard bacteriological tests, 308 of the 628 specimens were positive and 320 were negative. Results The sensitivity, specificity, positive predict value, negative predict value and Youden index on the 1009 specimen detection by the Trustline TB IgG/IgM rapid test kit was 61.3%, 79.8%, 83.3%, 55.6% and 0.411 respectively, and 53.7%, 89.0%, 88.9%, 53.8% and 0.426 by the control test kit. The differences in sensitivity and specificity between the two tests were statistically significant (P0.01). Further analysis showed that the detection rate of TrustlIne IgG/IgM test kit was 77.6% for positive specimens and 44.7% for negative specimens, while the related detection rates of the control test kit were 67.9% and 40.0% respectively; there was significant difference in positive specimen detection between the two kits. In addition, this study showed that 35 of the 1009 specimens were IgM positive by Trustline test kit, in which 30 (4.8%) were from TB patients, and 5 (1.3%) were from non TB individuals. Conclusion Trustline TB IgG/IgM rapid test kit is more sensitive in the detection of TB antibody in serum specimens compared with control test kit. The Trusline TB IgG/IgM test kit can detect both IgG and IgM in one test. It is recommended to be sued as TB screening test kit.