Syphilis is a sexually transmitted disease caused by Treponema pallidum subsp. pallidum; it can be effectively treated with penicillin yet remains prevalent worldwide, due in part to the shortcomings of current diagnostic tests. Here we report the production of soluble recombinant versions of three novel diagnostic candidate proteins, Tp0326, Tp0453, and a Tp0453-Tp0326 chimera. The sensitivities of these recombinant proteins were assessed by screening characterized serum samples from primary, secondary, and latent stages of infection (n = 169). The specificities were assessed by screening false positives identified with the standard diagnostic testing algorithm (n = 21), samples from patients with potentially cross-reactive infections (Leptospira spp., Borrelia burgdorferi, Helicobacter pylori, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, or cytomegalovirus) (n = 38), and samples from uninfected individuals (n = 11). The sensitivities of Tp0326, Tp0453, and the Tp0453-Tp0326 chimera were found to be 86%, 98%, and 98%, respectively, and the specificities were 99%, 100%, and 99%. In a direct comparison, the Captia syphilis (T. pallidum)-G enzyme immunoassay (Trinity Biotech) was used to screen the same serum samples and was found to have a sensitivity of 98% and a specificity of 90%. In particular, Tp0453 and the chimera exhibited superior accuracy in classifying analytical false-positive samples (100%, compared to 43% for the Captia assay). These findings identify Tp0453 and the Tp0453-Tp0326 chimera as novel syphilis-specific diagnostic candidates that surpass the performance of a currently available diagnostic enzyme immunoassay test for syphilis and that allow accurate detection of all stages of infection.