For a study, researchers determined that the DNA from many pathogens was detected in saliva. However, the presence and amount of Treponema pallidum DNA in patients with syphilis in saliva was unknown. About 234 patients with syphilis with different stages and 30 volunteers were registered. Paired saliva and plasma samples were collected from all patients. Saliva samples from 9 patients were collected every 4 hours, followed by treatment. Treponema pallidum DNA in samples was analyzed by nested polymerase chain reaction (PCR) and droplet digital PCR targeting polA and Tpp47. Treponema pallidum DNA detection rates in saliva and plasma came out to be 31.0% (9/29) and 51.7% (15/29) in primary syphilis (P=.11), 87.5% (63/72) and 61.1% (44/72) in secondary syphilis (P<.001), 25.6% (21/82) and 8.5% (7/82) in latent syphilis (P=.004), and 21.6% (11/51) and 5.9% (3/51) in symptomatic neurosyphilis (P=.02), respectively. Median (range) loads of Tpp47 and polA in saliva were 627 (0–101 200) and 726 (0–117 260) copies/mL, respectively, for patients with syphilis. In plasma, however, loads of Tpp47 and polA were found to be low: medians (range) of 0 (0–149.6) and 0 (0–176) copies/mL, respectively. The collection of saliva is convenient. The high loads of T. pallidum DNA in saliva and reduction after treatment indicated that saliva could not only be a diagnostic fluid for syphilis but also an indicator of therapeutic effectiveness.