Applying nanomaterials to highly sensitive, fast, and accurate detection of hazardous chemicals in water remains to be a great challenge. Herein, a Ti 3 C 2 -functionalized superlattice SERS substrate was developed, which can identify multiplex fish drug residues in pond water with a high sensitivity and reliable reproducibility. Specifically, a superlattice substrate was fabricated by vertically self-assembled bimetallic nanocuboids (BNCs) through the Marangoni effect, which exhibited an excellent SERS activity. Furthermore, the modification of the Ti 3 C 2 layer endows the substrate with both an increased SERS signal and an enhanced adsorption for multiple fish drugs, including crystal violet (CV), malachite green (MG), and methylene blue (MB). The limit of detection (LOD) was as low as 10 −12 M, while the signal fluctuation was as small as ∼5.9% between batches. More importantly, a trace amount of multiplex fish drug residues was detected in natural pond water, demonstrating a convincing practicality of such a Ti 3 C 2 -functionalized superlattice SERS substrate.