Surface-enhanced Raman spectroscopy (SERS) has been proven to be a promising analytical technique with sensitivity at the single-molecule level. However, one of the key problems preventing its real-world application lies in the great challenges that are encountered in the preparation of large-scale, reproducible, and highly sensitive SERS-active substrates. In this work, a new strategy is developed to fabricate an Ag collide SERS substrate by using cetyltrimethylammonium bromide (CTAB) as a connection agent. The developed SERS substrate can be developed on a large scale and is highly efficient, and it has high-density "hot spots" that enhance the yield enormously. We employed 4-methylbenzenethiol(4-MBT) as the SERS probe due to the strong Ag-S linkage. The SERS enhancement factor (EF) was calculated to be ~2.6 × 10. The efficacy of the proposed substrate is demonstrated for the detection of malachite green (MG) as an example. The limit of detection (LOD) for the MG assay is brought down to 1.0 × 10 M, and the relative standard deviation (RSD) for the intensity of the main Raman vibration modes (1620, 1038 cm) is less than 20%.