Abstract: In this study,a normalized TaqMan real-time polymerase chain reaction(PCR)approach was developed based on reference genes for quantitative detecting donkey milk adulterated with bovine milk in adulterated sterilized milk. With single-copy nuclear housekeeping genes replacing multicopy mitochondrial genes as reference and donkey-specific genes,an innovatively normalized assay based on a standard curve constructing by the Ct ratio(donkey genes/reference genes)vs. the donkey milk content was established with a good linear correlation(R²=0.9650)within the range of 5% to 100% of donkey milk. The quantification analysis of simulated adulterated milk samples containing 20%,50% and 80% of donkey milk revealed a high accuracy with an average recovery of 109.16% and an average CV value of 4.68%. Therefore,the method is rapid and accurate,which can determine whether donkey milk is adulterated and the proportion of adulteration.