Gold nanoparticles were homogeneously coated with silica using the silane coupling agent (3-aminopropyl)-trimethoxysilane to functionalize the gold surface and sodium silicate solution as the precursor of silica. The shell thickness could be well controlled by changing the amount of sodium silicate, reaction temperature and time. The Au@SiO2 core-shell nanoparticles with a suitable silica shell thickness exhibited optimal SERS activity and were self-assembled onto an ITO substrate in order to get a stable and reproducible SERS substrate. The conditions for preparing SERS substrates can be optimized by investigating the relationship between the intensity of SERS signals and the thickness of silica shell. The reproducible SERS measurements were performed by using 1,4-BDT and 4,4’-bipyridine as probe molecules. Within a certain concentration range, the linear relationship between the SERS intensities and the logarithm of concentration was obtained. The results revealed that the Au@SiO2 substrate assembled on ITO surface could be developed as a reproducible substrate for the quantitative analysis.