An Ag-coated polystyrene pellet hexagonal close packed array (Ag/PS HCA) substrate was prepared by L-B film method and magnetron sputtering technique. And Au@4MBA probes were obtained by linking the synthesized urchin-like Au nanoparticles with 4-mercaptobenzoic acid (4MBA). Then the single-stranded oligonucleotide DNA21 was linked with the substrate and the Au@4MBA probes, respectively, to construct the Au@4MBA-DNA21-Ag/PS HCA sandwich structure. After DNA21 and miRNA-21 hybridization, the DNA phosphodiester bond is cleaved using a double-strand specific cleavage enzyme (DSN), and finally SERS signal detection was performed. The experimental results show that the detection scheme of the tumor marker miRNA-21 based on the above-mentioned sandwich SERS structure and enzymatic cleavage technology has extremely high sensitivity and excellent specificity, and the detection limit reaches 0.853 fmol·L－1. In addition, compared with real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR), our scheme not only has the same results also has higher sensitivity.