To establish a method to detect Listeria monocytogenes(LMO) by PCR-pyrosequencing.MethodsA pair of PCR primers and a sequencing primer were designed according to the hly gene of LMO. The target gene was amplified by PCR specifically and single-stranded DNA templates for pyrosequencing were prepared from the PCR products. Finally, pyrosequencing was performed under the guidance of the sequencing primer. The strains were identified by aligning the sequencing results to the hly gene sequence in GenBank.Results PCR primers and sequencing primers showed good specificity. The results showed that a 249bp DNA fragment was amplified from 16LMO strains and the pyrosequencing results perfectly matched the hly gene sequence, while the control strains were both negative.ConclusionThis new established method is accurate and effective for rapid detection of LMO.