Surface-enhanced Raman Scattering (SERS) is an excellent analytical tool that can be used to develop assays and biosensors for the sensitive detection of small molecules in complex samples, taking advantage of plasmonic nanostructures. In this work, an aptamer was exploited to develop a competitive assay for Aflatoxin B1 (AFB1) by means of SERS analysis. After the careful optimization of the SERS-based assay, its potentiality for food safety assessment was demonstrated by detecting AFB1 in different extracts of AFB1-spiked slurry of hazelnut below the threshold defined by the regulatory limits.