Abstract: TaqMan real-time fluorescence PCR was used to rapidly identify and validate Listeria monocytogenes proficiency testing samples. According to the first method of GB 4789 series of standards, sample D15 and sample D16 in ACAS-PT526 proficiency testing were detected, and detected by traditional culture methods such as bacteria enrichment,separation, preliminary screening, and identification. Identification reagent API Listeria and automatic microbial identification system were also used in the identification process. At the same time, the enriched samples D15 and D16 and the culture of Listeria monocytogenes standard strain were rapidly identified by TaqMan real-time fluorescence PCR technology. The results showed that the sample D15 was identified as Listeria monocytogenes by API Listeria reagent, with a identification percentage of 98.6% and a T value of 1. And the automatic microbial identification system results indicated that the sample D15 was Listeria monocytogenes with a 99% possibility and sample D16 was Staphylococcus aureus with a99% possibility; Real-time fluorescence PCR identified that both the D15 LB1 enrichment culture and the single colony on the Listeria chromogenic medium plates had significant S-type amplification plots. Therefore, the real-time fluorescence PCR was consistent with the first experimental results of GB 4789.30—2016 standard, and the sample D15 was detected as Listeria monocytogenes, and the sample D16 was not detected as Listeria monocytogenes. TaqMan real-time fluorescence PCR method can be used for the rapid testing of microorganisms, especially for the auxiliary validation of blind samples.