Staphylococcus aureus (S. aureus) has been identified as a indicator of food contamination. In this study, a sensitive and accurate biosensor strategy for S. aureus through rolling circle amplification-assisted surface-enhanced Raman scattering (RCA-assisted-SERS), has been established. The work relies on the interaction between the aptamer and its partial complementary DNA strands fabricated on the surface of gold and silver-assisted magnetic microspheres and the subsequent detachment to trigger the activation of the RCA process. In RCA, template DNA, T4 DNA ligase, and Phi29 DNA polymerase were assembled to form long single-stranded DNA containing repetitive sequences. The gold core encapsulated with a layer of 4-nitrothiophenol and further covered with a silica shell was employed as the SERS nanoprobe (Au@NTP@SiO