In this Letter, a method for the rapid and efficient preparation of ultrasensitive detection substrates by assembling gold nanorod suspensions with the application of an alternating current (AC) field is proposed, and it is found that frequency and voltage are the effective means of regulation. A sandwich structure (parallel SiO2 plate) not only effectively slows down the evaporation rate, but also visually reveals the changes in the assembly process. Under the optimal assembly conditions, the sensitivity and uniformity of the substrate to different probe molecules are tested. The Raman detection results experimentally show that the detection limits of Rhodamine 6G (Rh6G), crystal violet (CV), and Aspartame (APM) molecular solutions are 10-14 M, 10-10 M, and 62.5 mg/L, respectively, and the mixed dye molecular solutions can also be effectively distinguished. Furthermore, Rh6G and CV characteristic peaks at 1647 cm-1 and 1619 cm-1 were measured at randomly selected positions, and their relative standard deviations (RSDs) were 5.63% and 8.45%, respectively, indicating that the substrate has good uniformity. The effective regulation of the self-assembly results of nanoparticles will further enhance the practical application effect of surface-enhanced Raman technology and expand the application prospects of this technology.