Abstract: Objective To establish a novel multiplex real-time fluorescent TaqMan PCR assay for the rapid detection of Listeria monocytogenes and Listeria ivanovii in simulated feces samples. Methods One pair of primers and TaqMan probes were respectively designed on hly gene in L. monocytogenes and smcL gene in L.ivanovii. Standard curve was produced. A real-time PCR assay to detect fecal samples containing L. monocytogenes and L. ivanovii was established. The specificity of the primers and probes was tested by using other Listeria spieces strains and other entero-pathogenic bacteria. With two enrichment steps, DNA from fecal samples contaminated artificially with different concentrations of L. monocytogenes and L. ivanovii were examined by using real-time PCR and traditional detection tests. Results Positive results were detected in the samples containing L. monocytogenes and L. ivanovii, but negative in the samples of other bacteria. The lowest detection limits of this assay were 2.45103 cfu/g for fecal sample with L.monocytogenes, and 2.92103 cfu/g for fecal sample with L. ivanovii. The whole process can be finished within 3 hours for fecal samples. Positive results were obtained in the samples with 6 cfu/g of L. monocytogenes and 5 cfu/g of L. ivanovii by using real-time PCR assay after sample enrichment. Conclusion The novel multiplex real-time TaqMan PCR for the detection of L. monocytogenes and L. ivanovii in simulated fecal samples showed high specificity and simplicity, and this simple and reliable assay can be used in the rapid detection of clinical samples to facilitate the survey of carriage or infection status of L. monocytogenes and L. ivanovii in population in China.