Objective To develop plasmid DNA reference materials that can be used for polymyxin antibiotic resistance encoding genes rapid detection.Methods The reference sequence of mcr-1， mcr-3 and mcr-5 were retrieved from the National Center for Biotechnology Information to construct recombinant plasmids and strains. The recombinant strains were subcultured for 15 generations to detect the genetic stability of the target gene. The recombinant plasmid was extracted and vacuum dried to prepare the standard sample. The limit of detection （LOD） of PCR and RT-qPCR was determined after the standard sample was hydrated and continuously diluted with 10-fold gradient， respectively. Multi-tube plasmid DNA standard samples were randomly selected to qualitatively and quantitatively evaluate the uniformity and storage stability at 4 ℃， 37 ℃ and -20 ℃ by PCR and UV spectrophotometer.Results Fragments of mcr-1， mcr-3 and mcr-5 that encoding polymyxin resistance mechanism were successfully obtained. Target genes in the recombinant strains could stably inherited after subcultured for 15 generations. The LOD of PCR and RT-qPCR of mcr-1， mcr-3 and mcr-5 standard samples were 1.67×10⁴ and 1.67 copies/μL， 1.31×10⁴ and 13.1 copies/μL， 1.55×10⁵ and 1.55 copies/μL， respectively. The mass F values of 12 standard samples randomly selected for each gene were all less than the F critical value， each gene could be detected by RT-qPCR and indicating that the uniformity of the samples met the requirements. When standard samples were stored at 4 ℃ for 90 d， 37 ℃ for 14 d and -20 ℃ for 360 d， the qualitative and quantitative test result showed it was stable without significant difference.Conclusion In this study， plasmid DNA standard samples that carrying mcr-1， mcr-3 and mcr-5 were prepared. The target genes could be stably inherited， the standard samples had good uniformity and storage stability， and could be used as a quality control samples for polymyxin antibiotics resistance encoding genes detection and mechanisms prediction.