Objective To develop a plasmid DNA reference material for rapid and comprehensive identification of Listeria monocytogenes. To develop a plasmid DNA reference material for rapid and comprehensive identification of Listeria monocytogenes.Methods The synthetic DNA fragment contained the hlyA, plcB and inlA, which were currently used for the detection of Listeria monocytogenes, was cloned into vector PUC57 to construct the plasmid reference material (pDNA Listeria). The quantity, homogeneity and stability of pDNA Listeria were evaluated by ultraviolet spectrophotometry (UV). real time quantitative polymerase chain reaction(qPCR) was used to evaluate the applicability of pDNA Listeria. The synthetic DNA fragment contains the hlyA, plcB, and inlA, which currently used for the detection of Listeria monocytogenes, was cloned into vector PUC57 to construct the plasmid reference material (pDNA Listeria ). The quantity, homogeneity and stability of pDNA Listeria were evaluated by ultraviolet spectrophotometry (UV). qPCR was used to evaluate the applicability of pDNA Listeria. Results The results showed that the fixed value of pDNA Listeria was 29.85 μg/mL,which had traceable values, reliable homogeneity and good stability.The pDNA Listeria could be staored at -20 ℃ for more than one year. The pDNA Listeria also provided comparable sensitivity and reliability to the genomic references material. The result showed that the fixed value of pDNA Listeria was 29.85 μg/mL,which had traceable values, reliable homogeneity and good stability.The pDNA Listeria could be staored at -20 ℃ for more than one year.The pDNA Listeria also provides comparable sensitivity and reliability to the genomic references material.Conclusion The pDNA Listeria could be used not only to identify Listeria monocytogenes but also to describe the biological characteristics such as virulence, pathogenicity, and invasiveness of Listeria monocytogenes at the same time. Importantly, the study proved the application of rapidly synthesized multiple targets plasmid serving as qPCR standard for pathogen identification. The pDNA Listeria could be used not only to identify Listeria monocytogenes but also to describe the biological characteristics such as virulence, pathogenicity, and invasiveness of Listeria monocytogenes at the same time. Importantly, the study proves the usefulness of rapidly synthesized multiple targets plasmid serving as qPCR standards for pathogen identification.