Pre-synthesized spherical Ag nanoparticles are firstly adsorbed onto silica spheres through bifunctional linking molecule, the silane reagent 3-aminopropyltrimethoxysilane, to form thin silver shells. These monodispersed Ag-coated silica nanospheres are assembled onto the glass substrates, which have been functionalized by 3-aminopropyltrimethoxysilane, to form close-packed three-dimensional Ag-coated silica nanosphere arrays by electrostatic interaction. The Ag-coated silica nanospheres were investigated as substrates for surface-enhanced Raman scattering using Rhodamine 6G as a probe molecule, and the enhancement factor of the Raman signal obtained on the Ag-coated silica nanospheres is about 1.74 × 109 for R6G. Compared with pure silver hydrosols, the prepared Ag-coated silica nanospheres have a higher Raman enhancing effect. The hybrid nanosphere substrates can be used as a highly sensitive chemical and biological sensor for Rhodamine 6G dye.