To establish a multiplex PCR method and to apply multiplex PCR for simultaneous detection of L. monocytogenes, S. flexneri and E. coli O157 in milk. According to DNA sequences of the hly gene of L. monoytogenes, ipaH of Shigella and the HlyA gene of E. coli O157:H7, three pairs of specific primers were screened and designed to compose optimized system and conditions of multiplex PCR. The three expected sizes were 360bp for L. monocytogenes, 512bp for Shigella and 240bp for E. coli O157:H7. The detection sensitivity of multiplex PCR were above 10² cfu/ mL in raw milk. The multiplex PCR took 6-8 hours to detect a sample. The results of the experiments demonstrated that the multiplex PCR assay was rapid, simple, sensitive and specific, which established important foundation for simultaneous detection for these three bacteria in raw milk.