In this paper, we present a convenient and economical method to fabricate a silver (Ag)-film semi-coated polystyrene (PS) nanosphere array substrate for surface-enhanced Raman spectroscopy (SERS). The SERS substrate was fabricated using the modified self-assembled method combined with the vacuum thermal evaporation method. By changing the thickness of the Ag film, the surface morphology of the Ag film coated on the PS nanospheres can be adjusted to obtain the optimized localized surface plasmonic resonance (LSPR) effect. The 3D-finite-difference time-domain simulation results show that the SERS substrate with an Ag film thickness of 10 nm has tens of times the electric field intensity enhancement. The Raman examination results show that the SERS substrate has excellent reliability and sensitivity using rhodamine-6G (R6G) and rhodamine-B (RB) as target analytes, and the Raman sensitivity can reach 10−10 M. Meanwhile, the SERS substrate has excellent uniformity based on the Raman mapping result. The Raman enhancement factor of the SERS substrate was estimated to be 5.1 × 106. This kind of fabrication method for the SERS substrate may be used in some applications of Raman examination.