Abstract: Objective To establish a dual TaqMan fluorescent quantitative polymerase chain reaction (qPCR) assay to detect Salmonella typhi and Salmonella paratyphi A in stool samples. Methods A set of primers and probes were designed and modified based on the sequence of STY1633 for S. typhi and SPA4289 for S. paratyphi A to establish a dual TaqMan fluorescent qPCR assay, and the specificity and sensitivity of the assay with DNA as template were evaluated and verified by detecting cultured 44 S. typhi strains, 30 S. paratyphi A strains, 88 strains of different serotypes Salmonella spp, non-Salmonella strains causing diarrhea and 8 species of bacteria causing bacteremia with fever as main symptom. The detection limit was determined by the simulated stool specimen supplemented with S. typhi and S. paratyphi A. Further verifying of specificity and sensitivity of the assay was performed with clinical stool samples from patients with fever and/or diarrheal symptoms. Results Totally 44 S. typhi isolates and 30 S. paratyphi A strains were detected to be positive with the established dual TaqMan fluorescent qPCR assay. The amplification of the remaining isolates, including main serotypes non-salmonella typhi strains, 5 species of enteric pathogen causing diarrhea and 8 species of bacteria causing bacteremia with fever as main symptom, were all negative. In the detection of purified total DNA from cultured S. typhi isolates, the detection limit of the assay was 1 pg per reaction (194 copies per reaction). The sensitivity achieved 2.5 pg per reaction (485 copies per reaction) in the extraction of nucleotide from cultured S. paratyphi A isolates. With the detection of crude nucleotide from S. typhi simulated stool, the detection limit reached 1 cfu/g and 102 cfu/g with and without enrichment of bacteria, respectively. The detection limit of the assay was 10cfu/g and 104cfu/g with and without enrichment of bacteria, respectively. For the stool samples from patients, the target gene amplification with the assay was positive for S. typhi and S. parayphi strains A and the detections were negative for the remaining 48 samples from diarrhea and/or fever patients infected with other pathogens. Conclusion The dual TaqMan fluorescent qPCR assay was established for simultaneous detection of S. typhi and S. paratyphi A with high sensitivity and specificity in stool sample, which would be suitable for rapid diagnosis of S. typhi and S. paratyphi A infection and identifying unknown pathogens causing fever, and would be helpful for early warning, intervention and treatment of typhoid fever by rapid detection and identification of pathogens.