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In this study, the second-generation high-throughput sequencing and DNA barcoding were combined to manually prepare multi-species mixed samples, and the mitochondrial gene CO І was used as a barcode to simultaneously identify the animal species in the mixed samples and identify endangered species. The results showed that under the family and genus level, the simultaneous detection rate of the species in the mixed samples was as high as 100%, and the species identification rate was as high as 89% at the species level, and with high sensitivity, as little as 1% of the trace species could be detected. However, nearly 30% of non-target classification annotations appeared at the species level. It can be concluded that the mini CO I barcoding can be applied to the simultaneous identification of animal species in mixed biological samples, and the species identification rate is high. Non-target classification match existing at the species level can be further improved by increasing the length of the barcoding, improving the sequencing technology, reference database and so on. In this study, DNA metabarcoding technology was used to evaluate the feasibility of identification of endangered animals in multi-species mixed biological samples with CO І, in order to lay a preliminary foundation for the advancement of DNA metabarcoding method in the field of wildlife forensic identification.