Raman spectroscopy can quickly achieve non-destructive, qualitative and quantitative detection, and analysis the molecular structure of substances. Herein, a facile and low-cost method for preparation of highly sensitivity SERS substrates was implemented through the displacement reaction of copper foam immersed in AgNO3 ethanol solution. Due to the 3D structure of copper film and homogenous displacement, the Ag–Cu substrate showed high performance SERS enhancement (1.25 × 107), and the lowest detection concentration for R6G reached 10–10 Mol/L. For glucose detection, mixed decanethiol (DT)/mercaptohexanol (MH) interlayer was used to enable glucose attach to the substrate surface, and the limit of detection reached to 1 uM/L. SERS substrate makes the Ag–Cu SERS substrate promising for biological applications.