We report a three-dimensional (3D) SERS substrate with different numbers of silver nanoparticle (Ag NP) layers using multilayer graphene oxide (GO) as a spacer. The SERS performance of the 3D nanostructure was investigated and it was found that the SERS effect increased as the number of Ag NP layers increased, and showed almost no change for more than four layers. We found that the SERS performance of the 3D nanostructures can be mainly attributed to the topmost hot spots which are closely related to the Ag NP layers in the 3D nanostructure. Furthermore, we explored 3D nanostructures with different Ag NP layers using the finite difference time domain method (FDTD). The 3D SERS substrates also exhibit excellent detection capability. The limit of detection (LOD) was calculated down to 10-15 M for R6G and 10-12 M for CV. In addition, the reproducibility of the 3D SERS substrate was attributed obviously to the increasing number of Ag NP layers. Based on these promising results, the highly sensitive detection of molecules such as malachite green was demonstrated for food safety inspection.