The invention relates to a MoS2/Cu nanometer particle SERS substrate and a preparing method thereof. The method comprises the following specific steps that a chip plated with a Cu film is evenly coated with an ammonium tetrathiomolybdate solution with a certain concentration in a spinning manner, and the novel MoS2/Cu nanometer particle SERS substrate is obtained through two times of continuous annealing processes. By means of the method, the technology is simple, batched preparation can be achieved, and a very good Raman enhancing effect can be provided while cost is saved; and actual application and popularization of the SERS substrate are greatly promoted.