The study was to establish a multiplex real-time quantitative PCR (qPCR) method based on TaqMan probes for simultaneous detection of Escherichia coli O157:H7， Listeria monocytogenes and Bacillus cereus. Specific primers and TaqMan probes were designed according to the conservative sequences of tir， mpl and entFM genes， corresponding E. coli O157:H7， L. monocytogenes and B. cereus， respectively， and a multiplex qPCR assay was presented to test their sensitivity， specificity and stability. The assay was used to detect pathogenic bacteria in contaminated milk samples， comparing with the national standard method. The experimental results showed that the multiplex qPCR assay exhibited high sensitivity， strong specificity and good stability， corresponding the minimum detection limited being 12 CFU/mL， only three kinds of target bacteria being amplified in the multiple-bacteria sample and the variation coefficient of repetitive amplification being less than 1%， respectively. The positive detection rate of bacteria-contaminated samples was up to 100% through the multiplex qPCR assay， consisting with the national standard method， and the testing can be fast finished within 6 h. It confirmed that the multiplex qPCR assay can rapidly and accurately detect the three common foodborne pathogens， namely E. coli O157:H7， L. monocytogenes and B. cereus in contaminated milk， and provided effective technical supports for food security.