The aim of this study is to establish a flow cytometry method based on fluorescent-labeled nucleic acidaptamer for detection of Staphylococcus aureus. By optimizing flow cytometry parameters, the amount and foldingmethods of the aptamer and bacterial treatment, specificity and sensitivity of this method was finally verified. The re-sults showed that aptamer which was not folding by the high temperature denaturation process has a good identifica-tion and the strongest binding with viable bacteria, the optimal working concentrations of aptamer is 250 nmol/L.We determined Staphylococcus aureus after pre-incubation in a total analysis time of 40 min, even in the presenceof Escherichia coli cells. The results proved that flow cytometry combined with fluorescently labeled aptamer offersadvantages of speed and specificity for the detection of Staphylococcus aureus.