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Surface-enhanced Raman scattering (SERS) spectra, which can provide large information about trace amount of chemical and biological species have been widely performed as a well-established tool in complex biological system. In this work, coomassie brilliant blue (R-250) with high affinity to proteins and high Raman activity was employed as a Raman reporter to probe prion protein (PrP) through a dual-aptamer mechanism, and thus an original strategy for PrP determination was proposed, which showed great potential to turn on the SERS response through specific recognition of anti-prion aptamers towards the target protein. Aptamers (Apt1 and Apt 2) recognizing distinct epitopes of PrP with high affinity were first conjugated to Ag@Si NPs, and Ag@Si-PrP/R-250-Ag@Si conjugates were obtained in the presence of PrP/R-250, inducing dramatically enhanced Raman signal. SERS responses enhanced with increasing amount of PrP and a linear equation of I SERS =6729.7+3091.2 c PrP was obtained in the range of 3.0–12.0×10 −9 M with the determination coefficient of 0.988. The proposed strategy is simple, rapid, and high specificity to probe protein–aptamer recognition in the solution. [ABSTRACT FROM AUTHOR]