Surface-enhanced Raman spectroscopy (SERS) can provide a fingerprint of molecules with ultrahigh sensitivity, down to the single-molecule level. However, accurate quantitative detection in practical samples using SERS is still a great challenge. Herein, a highly uniform shell-isolated Ag@SiO2 nanoparticle (Ag SHIN) monolayer film was prepared and employed as a substrate for the quantitative determination of melamine in milk samples through shell-isolated nanoparticle-enhanced Raman spectroscopy. The Ag SHIN film exhibits excellent reproducibility, high stability, as well as ultrahigh sensitivity with a limit of detection of 0.03 ppm. A linear relationship between the Raman intensity and the melamine concentration in a wide range (1 ppb to 5 ppm) was obtained. These results demonstrate that the Ag SHIN monolayer film is a promising and reliable substrate for the quantitative SERS analysis of practical samples. Copyright © 2017 John Wiley & Sons, Ltd.