A highly structured SERS biosensor was proposed for Staphylococcus aureus (S. aureus, ATCC 29213) detection. Polydimethylsiloxane (PDMS) film was chemically modified and gold and silver core–shell nanoparticles (Au@Ag NPs) were self-assembled on it. Sulfhydryl-modified aptamer of S. aureus was immobilized on the SERS substrate to form a capture substrate and the aptamer modified with 6-carboxy-x-rhodamine (ROX) was used as a signal molecular probe. The aptamer and target constructed a sandwich structure “capturing substrate-target-signal probe”. Concurrently, the bacterial surface was covered with Au@Ag NPs which resulted in more “hot spots” and improved the detection sensitivity. The SERS intensity was plotted against the logarithmic concentration of S. aureus (3.6 × 10 to 3.6 × 108 cfu/mL) and achieved an ultra-low detection response of 16 cfu/mL. S. aureus was detected in tap water, milk and fish samples with satisfactory recoveries (93.25%∼106.42%) and validation results (p > 0.05). Significantly, the method offered a speedy and accurate S. aureus examining in food/environmental settings.