Surface-enhanced Raman scattering (SERS) has become an advanced spectroscopic analysis method in the fields of chemistry, biomedical sensing, and imaging, owing to its excellent vibrational signal recognition and sensitivity for single-molecule detection. The effectiveness of SERS technology relies on the development of high-performance substrates, which must possess high sensitivity, uniformity, and repeatability. In this study, the enhanced substrates with high Raman activity were successfully prepared by adopting the three-phase self-assembly method to assemble gold nanorods (AuNRs) into nanopores of ultrathin porous alumina (AAO) films. By precisely controlling the pore size of AAO and the dimensions of AuNRs, the ability of the substrates and the Raman detection limits are enhanced significantly. Probe molecules, including Rhodamine 6G (R6G), Crystal Violet (CV), and Aspartame (APM), were selected to test the substrates sensitivity and uniformity, with detection limits of 10