Abstract: Hly gene of Listeria monocytogenes was using as the target gene, a pair of degenerate primers were designed with Primer 5.0, and the method of real-time fluorogenic quantitative PCR assay to detection of Listeria monocytogenes was established. The specificity and sensitivity of plasmid standard and different strains were verified, its preliminary application in Artificial inoculation fresh-cut melon was realized. The results showed that the method had good specificity and sensitivity, and sensitivity for plasmid standard was 7.69 ×10copies/μL, the minimum detection limit of artificial inoculation fresh-cut melon was 3.11×10cfu/mL.