The invention discloses an SERS substrate based on an REBCO template and a preparation method of the SERS substrate. The SERS substrate comprises an REBCO template layer and a metal modification layer, wherein the outer surface of the REBCO template layer is plated with the metal modification layer. The thickness of the REBCO template layer is continuously adjustable between 10 nm and 2,000 nm, and the structure is from an amorphous structure to a polycrystalline structure and can be freely adjusted in the a-axis and b-axis orientation and the c-axis orientation or the mixed orientation. The metal modification layer is a metal thin film or a metal particle layer. The thickness of the metal modification layer ranges from 5 nm to 200 nm. Compared with the prior art, the substrate and the preparation method of the substrate have the beneficial effects that the SERS substrate prepared through the method is good in uniformity and high in sensitivity, has the metal flexibility character and can meet multiple application needs; the REBCO growth mechanism and growth method are greatly studied, and industrial production under precise control can be achieved easily; and meanwhile the preparation method is simple, experiment parameters in the growth process are easier to control than those in a chemical method, and the industrial preparation cost can be lower than 0.5 dollar/cm<2>.