Surface enhanced Raman spectroscopy (SERS) can provide rich molecular structure information about ultra-sensitive, non-destructive, and rapid detection, with accuracy down to the single-molecule level. It has been widely applied to physics, chemistry, biomedicine, environmental science, materials science and other fields. Combining the advantages of metals and two-dimensional (2D) nanomaterials, various 2D metal composite structures have been proposed for SERS. However, the contribution of 2D nanomaterials in Raman enhancement is often limited. In this work, vertically aligned MoS₂ nanosheet composite with silver nanoparticles (Ag NPs) is proposed for SERS detection. Large-area vertically aligned MoS₂ nanosheets, which are grown directly on molybdenum (Mo) foil by using hydrothermal method, can effectively enhance molecular adsorption, light absorption, and provide dual electromagnetic and chemical enhancement. Furthermore, annealing treatment of the MoS₂ nanosheets significantly improves the efficiency of charge transfer between Ag NPs and MoS₂, thereby increasing the chemical contribution to SERS. The results demonstrate that the annealed MoS₂/Ag substrate exhibits outstanding SERS performance, with a detection limit for R6G molecules as low as 10^–12 mol/L, which is four orders of magnitude lower than that of the unannealed substrate. The enhancement factor (EF) is calculated to be approximately 1.08×10⁹, approaching the sensitivity required for single-molecule detection. Additionally, the substrate has high signal reproducibility at low concentrations, enabling ultra-sensitive detection of pesticide residues in aquatic products.