Abstract: Objective To establish a TaqMan-based multiplex real-time PCR assay for the detections of Salmonella and Listeria monocytogenes and conduct Salmonella and Listeria monocytogenes detections in food samples. Methods The specific primers and probes were designed in the conserved region of the invA gene for Salmonella and in the hlyA gene for Listeria monocytogenes, respectively.The reaction conditions were optimized, and the sensitivity,specificity and the stability of the assay were evaluated.The food samples collected from the supermarket were detected by this assay. Results The results showed that the assay had high specificity for the detections of Salmonella and Listeria monocytogenes without any evident cross-reaction with other pathogens such as Escherichia coli, Vibrio parahemolyticus and Shigella. The detection limits of the assay for Salmonella and Listeria monocytogenes were up to 10 cfu/ml and 100 cfu/ml respectively. Analysis with 105cfu/ml to 102cfu/ml Salmonella and Listeria monocytogenes samples demonstrated the high reproducibility with a coefficient of variation (CV) less than 5%. Compared with traditional culture method or monoplex real-time assay, this assay had significant higher sensitivity in the detection of 80 food samples for Salmonella and Listeria monocytogenes. The detection process could be finished within 10 hours, including 6 hours for enrichment. Conclusion The multiplex real-time RT-PCR assay could provide rapid, sensitive and reliable detections of Salmonella and Listeria monocytogenes and could be used in the detection of foodborne disease pathogens.