To investigate the detection threshold of Treponema pallidum specific antibody method by chemiluminescent immunoassay (CLIA) in Siemens ADVIA Centaur XP for Syphilis serological test, and compare with the results derived from CMIA, TP-WB and TPPA method. The result can serve as reference for the application of CLIA. In total 30 887 samples screened by Treponema pallidum specific antibody method were collected by Abbott architect i2000 CMIA from July 2018 to July 2019 in Yanda Hospital of Hebei Province. We selected 153 patients with the ratio of sample absorbance to critical value (S/CO) of 1–9 by CMIA screening of Treponema pallidum specific antibody as the research objects. The reverse sequence of syphilis serological detection was adopted, and TP-WB and TPPA were used as the confirmation methods respectively. MedCalc software was used to analyze the results of ROC curve, and the cut-off value was obtained. Chi square test was used to test the difference significance of counting data. The detection results of Treponema pallidum specific antibody in the same batch of serum samples were unequal by different methods. There was no significant difference between CLIA method and TPPA method, but significant difference between CLIA method with TP-WB method and CMIA method was found. TPPA test results and TP-WB test results were taken as gold standards, ROC curve analysis showed that the best diagnostic cutoff value of CLIA method was 4.01 and 16.06, respectively, and the area under the curve was 0.961 and 0.838. The suggested cutoff value of CLIA method is quite different when using different syphilis serological test methods as the gold standard, Therefore, when the S/CO value determined by CLIA is between 1.00 to 16.06, TP-WB method should be recommended as the first choice in laboratory serological test for recheck and confirmation to avoid clinical misdiagnosis.