Abstract: Objective To establish a real-time fluorescent quantitative polymerase chain reaction (qPCR) assay to detect Salmonella typhi in stool samples. Methods Primers were designed based on the sequence of STY1633, specific gene of S. typhi, for qPCR assay. The specificity and sensitivity of the qPCR assay established were evaluated and verified by detecting 92 strains of common pathogens other than S. typhi and 44 S. typhi strains. The detection limit was determined by using simulative stool specimen supplemented with S. typhi. Further verifying of the specificity and sensitivity of the qPCR assay was performed with clinical stool samples from patients with fever and /or diarrheal symptoms. Results Total 44 S. typhi isolates were amplified to be positive by this qPCR assay. Five enteric pathogens other than S. typhi causing diarrhea and 8 enteric non-enteric bacteria causing bacteremia with fever symptom were detected to be negative. For purified total DNA from pure culture isolates, the detection limit of the qPCR assay was 500 fg per reaction, equal to 97 copies per reaction. The limit of detection was 104 cfu/g and 50 cfu/g respectively with crude nucleotide from simulative stool samples before and after enrichment. Conclusion The qPCR assay for detecting S. typhi in stool sample with high sensitivity and specificity was established, which could be used in the rapid diagnosis of S. typhi infection and differential diagnosis of other pathogen infections with fever and diarrheal symptoms as well as in the early warning, prevention and control for typhoid fever.