The degenerate primers were designed to establish a PCR assay for synchronous and fast detection of staphylococcal enterotoxin A and B. According to the sequences of SEA and SEB gene, primers were designed and used for PCR. The lengths of the amplified primers were 105 bp and 135 bp. To evaluate the specificity of PCR assay, 2 strains of SEA, SEB and 4 strains of non-SEA-B were tested by PCR. SEB was 10-fold serially diluted and amplified by PCR to verify its sensitivity. The results showed that the detection of Staphylococcus aureus enterotoxin A, B strain DNA results were positive and 4 strains of control strains tested negative. The specificity of PCR products was confirmed by DNA sequencing. In addition, the minimum detection limit of DNA was 3.58 ng of SEB and the whole process was less than 20 h. A rapid, specific and sensitive detection method was established and the method can simultaneous detect Staphylococcal enterotoxins A, B in the same reaction condition.