Abstract: Objective:To establish a specific multiplex PCR system for the detection of beef,pork and chicken based on the polymorphic site of the animal mitochondrial cytb gene. Methods:The genomic DNA of meat was extracted and specific primers were designed using SNP sites of mtDNA cytb gene sequences of different species. Multiple PCR amplification was carried out to detect adulterated animal-derived components commonly found in beef by using different sizes of amplified product fragments. The minimum detection amount was determined by a sensitivity test. Results:The primers of the experimental design had good specificity. In the same reaction system and the same annealing temperature of 52 ℃,the beef DNA amplified to produce 149 bp specific bands,the pork DNA amplified fragment was 261 bp,the chicken DNA amplified fragment was 554 bp,and no non-specific amplification occurred. And the lowest concentration detected reaches the 100 pg/μL,and had a high sensitivity and applicability. Conclusion:According to the differential sites of animal mitochondrial cytb gene,a multiplex PCR system was developed to simultaneously detect beef,pork and chicken at one time. It can quickly,sensitively and efficiently analyze the source of adulterated components in food.