Abstract: Objective To establish the isolation and identification assay of Listeria monocytogenes in fecal sample, increase the detection rate and investigate the infection rate of Listeria monocytogenes in population in China. Methods Real-time PCR and traditional detections of Listeria monocytogenes were conducted after tow enrichments of artificial fecal samples, Results When Listeria monocytogenes concentration was 7 CFU /g in artificial fecal sample, the real-time PCR and culture results were positive. Conclusion The result of this study provides laboratory evidence for the isolation and identification of Listeria monocytogenes from fecal samples and etiological survey of food poisoning caused by Listeria monocytogenes, which would facilitate the survey of carriage or infection status of Listeria monocytogenes in population in China.