Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an amplification reaction using RNA as a template for detection. A real-time fluorescent RT-LAMP method for the detection ofStaphylococcus aureuswas established by optimizing primers designed for specific conserved nuc gene and optimizing the reaction conditions. The strain-specific and the sensitivity of pure culture ofStaphylococcus aureusand artificial contamination of skim milk samples were detected by this method. The RT-LAMP amplification reaction was completed at 65°C for 30minutes. The method had a strong strain specificity, the detection limit of pureStaphylococcus aureuswas 20 fg/μL and the sensitivity was 230 CFU/g in the detection of artificial contaminated skim milk samples. The established real-time fluorescent RT-LAMP method can provide a powerful means for rapid detection ofStaphylococcus aureusin the field.