In this study, a three-dimensional (3D) Cu(OH)₂-Ag substrate with abundant “hotspots” is developed. First, the 3D Cu(OH)₂ nanotubes (NTs) are developed on the surface of copper sheets through chemical etching. Then, silver nanoparticles (NPs) are adsorbed onto the surface of Cu(OH)₂ NTs using self-assembly technique. The prepared Cu(OH)₂-Ag substrate has a high Raman sensitivity with a mass concentration limit of detection (LOD) of 8.9×10 ^-10 g/L for probe molecule rhodamine 6G (R6G). Additionally, it has high uniformity and reproducibility with a relative standard deviation (RSD) of 8.79% and 3.49%, respectively. Simultaneously, the substrate has excellent sensitivity to malachite green (MG), and the mass concentration LOD of MG in river water is 2.13×10 ^-8 g/L. The 3D substrate is easy to fabricate and has excellent surface-enhanced Raman spectroscopy (SERS) performances. It shows that the substrate has great application prospects in detecting illegal fishery drugs in water.