Abstract: The present disclosure herein discloses a preparation method of a polyurethane-based nano-silver SERS substrate, belongs to the technical field of Raman spectrums, and aims to solve problems of complex preparation process, low sensitivity and the like of SERS substrates. The method uses solidified polyurethane as a skeleton, and the polyurethane adsorbs nano silver particles onto its surface due to a porous surface structure and adsorptivity, so an SERS substrate with crystal violet as a probe molecule and having a limit of detection as low as 10?10 M is obtained. The SERS substrate prepared by the method has a large surface area, adsorbs a large number of target molecules, and is easy to prepare, high in sensitivity, and conducive to qualitative and quantitative analysis of SERS.