This work is devoted to revealing the parameters of preparing SERS substrates for studying a low concentration trimethylamine oxide (TMAO) solution by Raman spectroscopy. A study was made of the effect of treatment with a 3,5% HCl solution, deionized water, and isopropanol vapor of planar surface enhance Raman spectroscopy (SERS) structures on the resulting Raman spectra of TMAO. We used a SERS substrate representing a multilayer structure: a mirror silver layer, a thin dielectric SiO2 insulating layer, and an array of plasmonic Ag nanoparticles about 25 nm in size. The influence of the duration of the substrate soaking in the analyte solution on the quality of the Raman spectra was established. Raman studies of SERS substrates with TMAO were carried out using 532 and 785 nm lasers.