Staphylococcus aureus has been frequently reported as an agent leading to outbreaks of disease in raw milk. The conventional quantitative PCR (qPCR) are unable to differentiate DNA of viable Staph. aureus from dead ones. The aim of this study was to use sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) coupled with lysostaphin to detect viable Staph. aureus. The cell suspensions were treated with SDS and PMA before DNA extraction, because SDS could enhance DNA intercalating ability to dead cells of PMA. The lysostaphin was applied to improve the effectiveness of DNA extraction. The reliability and specificity of this method were further determined by the detection of Staph. aureus in spiked milk. The results showed that there were significant differences between the SDS-PMA-qPCR and qPCR when a final concentration of 200μg/mL of lysostaphin was added in DNA extraction (P < 0.001). A standard curve with a good linear relationship (R² = 0.9983, amplification efficiency = 96%) was obtained when lysostaphin was applied at a final concentration of 200 μg/mL. The viable Staph. aureus could be effectively detected when SDS and PMA concentrations were 100 ppm and 40 μM, respectively. Compared with conventional qPCR, the SDS–PMA–qPCR assay coupled with lysostaphin was more specific and sensitive. Therefore, this method could detect the number of viable Staph. aureus accurately.