Abstract: According to the multiple hly gene sequences of Listeria monocytogenes, a pair of degenerate primers and a probe was designed, and the method of real-time fluorogenic quantitative PCR assay to detection of Listeria monocytogenes was established, the specificity and sensitivity were verified; its preliminary application in artificial inoculation fresh-cut melon was realized.The results showed that the method had good specificity and sensitivity for plasmid standard was 1.12 × 102 copies /μL, the minimum detection limit of artificial inoculation fresh-cut melon was 6.28 × 102 cfu /mL. This method can lay a technology foundation to detect and control the pathogenic microorganism of fresh-cut fruits and vegetables.