Abstract: According to the mitochondrial cytochrome-b sequences of pig, cattle and sheep in the Genebank, we designed universal forward primer and species-specific reversal primer. In order to get the multiplex PCR optimum condition, different proportion of primers, PCR conditions and template volume were established. The result showed that the method could amplify sample contend pig, cattle and sheep, and the minimal determination concentration scope of this method was 10% of adulteration level. This method was used in the Chengdu market investigation for pig, cattle and sheep which could get a good effect.