Human serum albumin (HSA) is an important protein in human blood plasma and is commonly used in assays such as spectrophotometry, fluorescence and resonance Rayleigh scattering (RRS); however, its detection using SERS quantitative analysis methods is rarely reported. In this study, the as-prepared nanogold (NG) particles were aggregated to stable NG aggregates (NGA) with a strong resonance Rayleigh scattering effect in pH 6.6 phosphate buffer solution containing NaCl. On addition of Coomassie brilliant blue G-250 (CBB) as a molecular probe, it was absorbed on the surface of the NGA, which exhibited the strongest surface-enhanced Raman scattering (SERS) peak at 1171 cm⁻¹, and 2.9 × 10⁻⁸–4.68 × 10⁻⁷ mol L⁻¹ of CBB in aqueous solutions can be detected directly using the SERS sensor platform. Using this sensor platform to monitor the concentration changes of CBB in the SERS quenching reaction of HSA–CBB, a new simple and sensitive SERS method was developed for the quantitative analysis of 0.04–2.0 μg mL⁻¹ HSA, which could be utilized to study the interaction of HSA with a dye.