The invention discloses a detection kit for quickly identifying donkey skin, horse skin and mule skin, which is used for accurately identifying donkeys, horses, mules and jennets by creatively applying 16SrRNA genes of CKM (creatine kinase) karyogene (nDNA) and mitochondrial genome DNA (mtDNA) at the same time from genetic background of the horses, the donkeys and the mules. The kit adopts an molecular beacon probe (MB) method to carry out a quintuple multicolor fluorescence quantitative PCR (polymerase chain reaction) detection technology, and also can detect donkey, horse, mule and jennet components; and moreover, the kit is added with exogenous internal reference as an interior label, can detect and avoid false negative results produced by PCR inhibitor contained in a sample. The quintuple multicolor fluorescence quantitative PCR is used for detecting in the same tube without opening a cover, so that the detection kit is not easy to pollute, and is accurate and stable, simple to operate, extremely high in sensitivity, strong in specificity, and the like, and therefore, a novel way is explored for identifying the donkey skin, horse skin and mule skin.