Neurosyphilis (NS) is a central nervous system infection caused by Treponema pallidum subspecies pallidum. NS has been associated with asymptomatic meningitis, which promotes severe and irreversible organ dysfunction and damage. Therefore, investigating NS pathogenesis is of great importance as this will likely reduce the incidence of sequelae. Besides, diagnosis of NS is quite challenging for clinicians due to lack of gold standard tests for diagnosing NS modalities. Here, using proteomic and machine learning model techniques, we characterize 223 cerebrospinal fluid (CSF) to identify the diagnostic markers of NS and provide mechanistic insights on associated inflammatory responses. Three biomarkers were of diagnostic value for NS through multicenter verification of additional 115 CSF samples. Through proteomics and single cell transcriptome analysis of brain tissue and CSF from NS patients, combined with brain organoid model and histopathological analysis, we found that inflammatory injury in syphilis is predominantly mediated through the Toll-like/NF-κB signal pathway. We anticipate that the biomarkers identified will become an effective means for assisting diagnosis of NS in the future. Our insights into the NS pathogenesis in brain tissue can be useful for discovering therapeutics or benefit drug target discovery for NS patients.