The invention belongs to the technical field of nano-imprinting and spectra, and in particular relates to a multilayer structure surface enhanced Raman scattering base and a preparation method thereof. The surface enhanced Raman scattering base is simple in process, high in efficiency, high in enhancement factor and biocompatible. The base consists of a substrate and a periodic nano columnar structure which is positioned on the substrate, wherein the nano columnar structure is a multilayer structure; the multilayer structure consists of alternation layers and a gold layer; the alternation layers consist of silver and media; the gold layer is positioned on the topmost layer. A nano-imprinting technology is used as a core technology, and a reactive ion etching process, a metal evaporation process, a silicon dioxide plating process, a metal peeling process and the like are combined to prepare a multilayer nano structure, so that the technical problems that a silver structure base is not biocompatible and a gold structure base is low in enhancement factor are solved; on the basis of ensuring biocompatibility, the enhancement factor of the base is greatly improved, the detection is efficient and sensitive, and the base can be applied to biological detection after further treatment.