This research focuses on establishment of a way that distinguish dead Staphylococcus aureus from the viable to avoid false-positive results caused by the ordinary DNA-based polymerase chain reaction in food detection.Primer and probe are designed by ourselves.One-step reverse transcription polymerase chain reaction(RT-PCR) is applied to detect mRNA of Staphylococcus aureus femA.Results show that only living Staphylococcus aureus are positive and deaths are negative.In pure culture,the coefficient of variation is 0.15 by repeat detection,and the analytical sensitivity and detection limit of the assay are 9 × 102 CFU/mL and 1 /3 CFU /3mL respectively.Experiments have shown that the one-step reverse transcription-polymerase chain reaction assay established in this study is not only a rapid,sensitive and specific method but also can effectively distinguish the dead from the viable Staphylococcus aureus.