In order to study the resuscitation of Staphylococcus aureus in the viable but non-culturable state (VBNC), three factors (temperature, salt concentrations and pH) were investigated to induce VBNC Staphylococcus aureus. Four different resuscitation methods were attempted and a new detection method using qPCR and PMA in combination with qPCR was established to detect Staphyloccocus aureus. The results showed that the VBNC samples could resuscitate in 48 h after disposed in 8% Tween80. In addition, PMA-qPCR was an effective method for detection of VBNC Staphyloccocus aureus, which efficiently overcome the shortcomings of plate counts.