Abstract: To detect Staphylococcus aureus enterotoxins D in milk, a real- time PCR assay based on Taq Man probe was developed with the specific primers and one Taq Man probe designed according to the target gene of SED of Staphylococcus aureus enterotoxins D and a recombinant plasmid containing the target gene constructed as a standard control. Rapid detection of Staphylococcus aureus enterotoxins D was achieved, and the evaluation of detection sensitivity, specificity and repeatability was carried out. This method was confirmed to have good specificity.The correlation coefficient of the standard curve was 0.999 and the sensitivity was 40 copies / m L. The detection limit of Staphylococcus aureus enterotoxins D was 1.0 × 102 CFU / m L in milk.The developed real- time PCR assay based on Taq Man probe for the detection of Staphylococcus aureus enterotoxins D was rapid, sensitive, specific, and could be further used in rapid screening of staphylococcal enterotoxin D, with good prospects.