Surface-enhanced Raman spectroscopy has emerged as a powerful spectroscopy technique for detection with its capacity for label-free, nondestructive analysis, and ultrasensitive characterization. High-performance surface-enhanced Raman scattering (SERS) substrates with homogeneity and low cost are the key factors in chemical and biomedical analysis. In this study, we propose the technique of atomic force microscopy (AFM) scratching and nanoskiving to prepare periodic folded gold (Au) nanostructures as SERS substrates. Initially, folded Au nanostructures with tunable nanogaps and periodic structures are created through the scratching of Au films by AFM, the deposition of Ag/ Au films, and the cutting of epoxy resin, reducing fabrication cost and operational complexity. Periodic folded Au nanostructures show the three-dimensional nanofocusing effect, hotspot effect, and standing wave effect to generate an extremely high electromagnetic field. As a typical molecule to be tested, p-aminothiophenol has the lowest detection limit of up to 10 −9 M, owing to the balance between the electromagnetic field energy concentration and the transmission loss in periodic folded Au nanostructures. Finally, by precisely controlling the periods and nanogap widths of the folded Au nanostructures, the synergistic effect of surface plasmon resonance is optimized and shows good SERS properties, providing a new strategy for the preparation of plasmonic nanostructures.