The enhanced Raman spectroscopy (SERS) substrates were obtained using self-assembly Ag nanoparticles on the surface of glass modified with 3-aminopropyltrimethoxysilane (APTES), where the Ag nanoparticles were synthesized by aqueous reduction of AgNO3 with trisodium citrate. The surface morphology of thin films with assembled silver nanoparticles was studied with UV-Vis spectroscopy and SEM. With the probe molecule of Rhodamine 6G, the Raman spectra on self-assembled Ag nanoparticle substrates were detected, which showed a great increase of Raman signals for Rh6. The surface plasmon resonance (SPR) of the assembled silver nanoparticles depended on the distance between the particles, the adsorption of molecules, and the assembly structure. Experimental results indicated that he intensities of the Raman peaks increased concomitantly with the increase in the concentration of Rh 6G. The detection limit of the assembled Ag nanostructures was 10−8M. In this paper, the results further confirmed that the nanoparticles assembled method was an effective method for the increase the signal of SERS.