Objective To develop a highly sensitive，low-cost and rapid assay method for the detection of Staphylococcus aureusenes. Method Combining F₀ F₁ -ATPase molecular motor with immunotechnology to establish a rapidly technology for detecting Staphylococcus aureusenes. F1300 is a fluorescence indicator sensitive to the change of pH. F1300 was labeled first on the inner surface of chromatophore，which is the carrier of F₀ F₁ -ATPase. A complex of Staphylococcus aureusenes antibody-biotin-streptavidin-biotin-β subunit monoclonal antibody was then connected to the β subunit of F₀ F₁ -ATPase to construct an immunobiosensor; then the Staphylococcus aureusenes can be captured by the antibody anchored on the complex. The activity of F₀ F₁ -ATPase changed with the content of Staphylococcus aureusenes loaded on the biosensor. The fluorescence of tagged F1300 was relative to the change of pH induced by the variation of F₀ F₁ -ATPase activities. Results The time taken by this examination was only 4. 5 h and a favorable gradient was shown at 10² － 10⁴ CFU /well. Conclusion The method was time saving in examination，simple，low-cost and sensitive and met the requirements of detecting technology.