Abstract: Disclosed are a method for preparing a AgNPs@SASP substrate material and application thereof. The present disclosure uses cyclohexane as an organic phase and ethanol as an inducer, and uses the interfacial tension of an organic/water interface for self-assembly to arrange the nanoparticles in a single layer. The addition of a low-dielectric solvent as an inducer reduces the charge density on the nanoparticles. As a result, the van der Waals force combined with the reduction of the Coulomb repulsion force cause the nanoparticles to reassemble at the organic/water interface to form a new equilibrium, and a SERS platform of AgNPs in a two-dimensional array is prepared. SERS detection and peak attribution analysis of different food-borne pathogen spores are carried out, and qualitative analysis is carried out by a multivariate statistical method to realize the rapid identification of spore-forming bacteria.