Abstract: Objective To develop a sensitive and specific real-time TaqMan polymerase chain reaction (PCR) assay for the detection of Plesiomonas shigelloides. Methods One set of primer and probe was designed based on the specific sequences of the 23S rRNA, and 7500 real-time PCR system was used to evaluate the sensitivity of the assay, and the specificity was evaluated by using 30 common enteropathogenic bacteria and some isolates causing nosocomial infection. Results The sensitivity of the real-time PCR assay was 1102 copies per reaction for testing recombinant plasmids and 310-2 pg per reaction for testing Plesiomonas shigelloides genome DNA. No specific amplifications were presented when the 30 common enteropathogenic bacteria and some isolates causing nosocomial infection were tested. Furthermore, the assay could be finished within 2 hours. Conclusion The real-time TaqMan PCR assay developed in our study was sensitive, specific and rapid, which could be used for the detection and isolation of Plesiomonas shigelloides.