Using porcine， bovine and bear mitochondrial cytochrome b genes，SDS-proteinase K cleavage（SDS-PK） was used to extract the bile DNA， Primer Premier 5.0 software was used to design the species-specific primers that could amplify the different sizes of DNA fragments without cross reaction. After the specific amplification， the amplified bands were cloned and sequenced， and compared with the registered sequences of GenBank database. The multiplex PCR was established and optimized， and the specificity and sensitivity were evaluated，and the method was used to identify 27 simulated mixed adulterated samples.

The established method for DNA extraction from pig， bovine，and bear could be completed within 2 h， and the DNA purities were 2.04， 1.69， and 1.70， respectively， and the concentrations were 158.63， 189.34，and 148.55 mg·L^－1. The species-specific primers were designed to amplify the pig， bovine， and bear. The sequencing results showed that the homology between the samples and the registered sequences from GenBank database was more than 99%. The primers of each species in the PCR system had strong specificity and no non-specific amplification. In triple PCR system，the primers did not interfere with each other；the detection sensitivity was high， and it could be successfully detected when the DNA concentration of any of the three target samples reduced to 1 mg·L^－1. The test results of 27 artificially simulated bear bile powder adulterated samples with different species and different proportions were consistent with the preset mixing conditions. The blind random sampling was repeated for 5 times， and the results were stable.

The method that can simultaneously identify the pig， bovine， and bear bile powder through a single experiment is successfully established， which is simple， sensitive and efficient， and can be applied to the identification of bear bile powder and its common animal orgin adulteration.