The invention provides a preparation method of a twist angle-controllable multilayer graphene structure, belonging to the technical field of material, and relates to the preparation of a double-layer or multilayer graphene structure with controllable twist angle(s) between or among layers. The method comprises the steps of firstly, cutting a signal layer of graphene single crystal on an SiO2/ Si substrate into two parts, and carrying out spin coating on polymethyl methacrylate (PMMA) on the two parts; after that, removing the SiO2 layer by corrosion, and enabling the two parts of PMMA films carrying the graphene to fall off; transferring one of the two parts of PMMA films onto a new SiO2/Si substrate, removing PMMA, and fixing the other part of PMMA film onto a glass sheet; respectively fixing the new substrate and the glass sheet; adjusting the angles and the positions of the two pieces of graphene under a micromainpulator system, and stacking the two pieces of graphene together; removing the PMMA to obtain the double-layer graphene structure with a certain twist angle; repeating the process to obtain the twist angle-controllable multilayer graphene structure. After the method is used, the angle-controllable multilayer graphene structure can be flexibly and conveniently obtained, and foundation is laid for the application of the graphene on a photoelectronic device.