In this study, a sensitive and robust single-particle SERS sensing platform for profiling of Staphylococcus aureus (SA) in complex samples was developed. The hierarchical Au microparticles (HAMs) with intense surface-enhanced Raman scattering (SERS) enhancement were synthesized. These HAMs were capable of capturing SA by modifying them with specific SA-targeting aptamers. The captured SA was then selectively bound to aptamer-modified spherical Au nanoparticles (AuNPs), resulting in a sandwiched structure of HAMs-bacteria-AuNPs. This structure led to enhanced SERS signals, enabling the quantitative detection of SA. We achieved a broad and linear range of SA detection from 1 × 10¹ to 1 × 10⁶ cfu/mL, with a limit of detection (LOD) of 0.93 cfu/mL. Furthermore, we successfully measured the presence of SA in milk samples, obtaining a recovery rate from 98.2% to 101.1%. These results demonstrate the potential application of the developed method in food safety, infectious diseases, and other health-related safety supervision.