PURPOSE: Syphilis is a sexually transmitted bacterial infection caused by , which can lead to chronic morbidity and adverse complications. In clinical practice, serofast status (SF) patients present with clinical symptoms that are very similar to those of healthy individuals or syphilis-cured patients, and often require prolonged follow-up for diagnosis. Currently, there is increasing interest in the potential of plasma exosome-derived miRNA as a biomarker for the detection of infectious diseases. In this study, we aimed to explore the diagnostic potential of miRNA in SF and its possible biological implications. PATIENTS AND METHODS: Exosome-derived miRNAs were isolated from peripheral plasma samples obtained from 20 patients with secondary syphilis (SS), SF, serologically cured syphilis (SC), and healthy controls (HC), and differentially expressed miRNAs (DEmiRNAs) were identified by microarray analysis. Prediction of potential target genes, functional annotation, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were then performed. The expression of selected miRNAs was confirmed in 37 patients by quantitative reverse transcription polymerase chain reaction (RT-qPCR). A receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic performance of these miRNAs in differentiating syphilis from HC or SC. RESULTS: The expression profile of plasma exosome-derived miRNA was discovered in individuals with SF through microarray analysis. The targeted genes of DEmiRNAs were found to be involved in diverse biological processes according to GO and KEGG analysis, such as regulation of transcription, mitochondria, Golgi, immune system, apoptosis, Ras signaling pathway, etc. Using RT-qPCR validation, miR-1273g-3p, miR-4485-5p, miR-197-3p, and miR-1908-3p showed significant upregulation in patients with SF. These miRNAs exhibited a superior diagnostic ability, either individually or combined, to distinguish SF from SC or HC. CONCLUSION: The DEmiRNAs in plasma exosomes may play a role in the pathogenesis of SF and have the potential to become a noble and effective diagnostic method.