To research surface-enhanced Raman scattering effect of different diameters’ polystyrene nanospheres (Ag-coated) substrates, we had prepared a new surface-enhanced Raman scattering substrate. It was prepared by using spin-coating and self-assembled to form monolayer and hexagonal close-packed (HCP) order colloidal-crystal films with PS nanospheres of different diameters. Then, a 30 nm Ag film was deposited on the PS arrays with magnetron sputtering. We detected Raman scattered spectrum by means of Roman spectroscopy instrument with Rhodamine R6G as a probe molecule, and the conclusion was drawn that all the substrates of Ag-coated PS nanospheres displayed stronger Raman enhancement. With the increase of the diameter of the microspheres, the degree of fluctuation of polystyrene nanospheres array was continuously strengthened (increasing roughness), and the SERS signal was enhanced gradually. The signal peak reached a maximum when the ball diameter was 600 nm, and then we aquired an optimal SERS substrate. Simultaneously, we found that the SERS spectra of R6G of a high signal to noise ratio was obtained on the substrate, and associated with the benzene ring within a range of CC double bond stretching vibration characteristic spectrum and the inner surface and the outer surface deformation vibration characteristic spectra were significantly enhanced. The single and large-scale Raman scattering substrate presents especial nanostructure of high and low distribution. The depth and the gap between different PS microspheres show the obvious differences, which can significantly improve the surface morphology structure of Ag film and Raman scattering effect of PS nanospheres substrate. The substrate presenting special arrays’ structure has significantly great potential in exploring molecule of chemical and biological filed.