Abstract: This study combined the second generation sequencing with DNA barcoding technology. The mixed samples were manually prepared, and the mitochondrial gene Cyt b was used as a barcode to simultaneously identify the species in the mixed samples. The results showed that at the genus and species level, the identification rate of species in the mixed samples was 89%, and the sensitivity was high, as low as 1% of the species components were detected at the same time, but the species discrimination rate was low, nearly 50% of the taxonomy matches were non-target matches. The conclusion of this study was that the mini Cyt b fragment could be used as a barcode for simu-ltaneous identification of animal species in mixed biological samples. The discrimination rate was high at the genus level. At the species level, there was a high detection and low discrimination rate. This problem will be further improved by increasing the length of barcode. In this study, DNA metabarcoding technology was used to evaluate the feasibility of Cyt b in identifying endangered animals in multi-species mixed biological samples, in order to lay a preliminary foundation for the advancement of DNA metabarcoding method in the field of wildlife forensic identification.