In this paper, we propose a method based on porous alumina (AAO) substrate as a template, using Alternating current (AC) electric field assembly technology to assemble large gold ball nanoparticles coated with CTAB in AAO nanopores, and analyze the effects of AC electric field frequency and voltage on the assembly results, and obtain the optimal conditions for the preparation of self-assembled single particles in the pores. On this basis, the prepared substrate was soaked in a colloidal solution of small gold balls modified with sodium citrate, and the single particles modified by NDT molecules were quantitatively coupled with a single morphology and coated with different nanoparticles on the surface to obtain Heterodimers@AAO array substrate. Rhodamine 6G, Crystal violet and Aspartame were selected as probe molecules to explore the high Raman activity and enhanced uniformity of the substrates, and the obtained results provided experimental support for the engineering application of Raman spectroscopy.