Abstract: In this study, a Dual-priming oligonucleotide (DPO) -based PCR method for specific detection of L.monocytogenes was developed with iap gene as target. Annealing temperature insensitivity, specificity and detection sensitivity of the DPO-PCR method were tested and its preliminary application was carried out in practice. Results showed that the detection sensitivity of the DPO-PCR method was 151CFU / mL. In the annealing temperature insensitivity test, compared with conventional PCR primers, DPO primers were able to efficiently amplify target gene in the annealing temperature range of 48⁶8℃. In the specificity test, the DPOPCR method showed a higher specificity for the target bacteria than conventional PCR method and no nonspecific amplification reactions were observed in DPO-PCR. In practice, 9 L.monocytogenes positive samples from 130 samples were detected by the DPO-PCR method, which was in accordance with the testing results according to GB/T 4789.30-2008, showing a better practicability. The DPO-PCR provided a new method for fast and accurate detection of L.monocytogenes.