The aim of this study was to optimize candidate antigen proteins for serological screening of Chlamydia trachomatis infection. C. trachomatis positive serum and swabs of genital secretions were collected from 50 patients in the Tianjin Medical University General Hospital, as well as from 30 patients negative for C. trachomatis. Samples were assessed by colloidal gold assay in a sexually transmitted disease clinic as follows: serum antibodies for eight kinds of C. trachomatis immunodominant proteins (Pgp3, CPAF, CT143, CT101, CT694, CT875, CT813, and IncA) were detected, and two traditional gold standards, immunofluorescence and C. trachomatis cell culture of genital secretions, were used for comparison in order to determine the antigen protein combinations with the highest sensitivity and specificity. Of the 50 samples that tested positive for C. trachomatis infection by colloidal gold assay, 44 tested positive by micro-immunofluorescence, whereas 6 tested negative. In contrast, 14 samples tested positive by cell culture, whereas 36 tested negative. Serological results of the immunodominant protein combination of Pgp3, CT694, and CT875 shared positive coincidence rates of 97.73 and 92.86% with C. trachomatis micro-immunofluorescence and cell culture, respectively. No antibodies of the three proteins were detected in the 30 C. trachomatis samples that tested negative by colloidal gold assay; these samples also tested negative in C. trachomatis genital secretion culture. Overall, the combination of the three immunodominant proteins Pgp3, CT694, and CT875 had good sensitivity and specificity for serological screening of C. trachomatis infection, and the process was simple and easy to apply.