In the study, fluorescent enzyme-linked immnoabsorbent assay for detection of Staphylococcus aureus was established with IgG from pig as capture antibody and quantum dot nanobeads (QBs) labeled vancomycin (QB-Vans) as testing antibody. Quantum dot of about 100 nm partical size nanobeads were prepared and linked with vancomycin. The optimum concentrations of salt ions were 0.01 mol/L, and the optimum pH was 6.0. Under the optimum conditions, the detection sensitivity for S. aureus was 104 CFU/mL, and there was no cross-reaction with other pathogenic bacteria. Thus, the method could be used for rapid screening of S. aureus, for the clinical monitoring and foodborne pathogens detection.