A rapid and sensitive SERS biosensor based on aptamer-modified Fe₃O₄@SiO₂@Ag and Au-MPBA/DTNB@Ag was constructed for simultaneous detection of Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli). This process can be simply described as a capture-enrichment-labeling (CEL) process. The monodisperse and strong magnetic Fe₃O₄@SiO₂@Ag-P1/P2 can quickly capture and enrich pathogenic bacteria. Fe₃O₄@SiO₂@Ag and Au@Ag synergistically enhanced the Raman signal of Raman reporters for labeling bacteria. Therefore, the dual-enhanced SERS biosensor can rapidly detect S. aureus and E. coli, and the limit of detection (LOD) is 1 CFU/mL, which is obviously superior to other SERS biosensors of the same type. It is proved to be effective in milk samples, and the total detection time is only 20 min. More importantly, when the aptamer is converted, the SERS biosensor can be used for other types of bacterial detection. The SERS biosensor based on dual-aptamer has the advantages of short analysis time, simple steps and high sensitivity.