The invention belongs to a preparation method of a biosensor, and relates to a preparation method of a graphene field-effect transistor biosensor. The preparation method comprises the following steps: (1) preparing a single-layer graphene copper plate; (2) preparing devices on a substrate layer of a sensor; (3) performing directional transfer on graphene; and (4) heating to bake the sensor, removing PMMA (polymethyl methacrylate) with acetone, performing vacuum annealing, and modifying the graphene with probes to obtain the graphene field-effect transistor biosensor. According to the preparation method provided by the invention, large-scale preparation of the biosensor based on CVD (chemical vapor deposition) graphene can be realized, the copper etching time is short, and the preparation method is simple and quick and can greatly lower the manufacturing cost; and in the preparation process, the PMMA polymer is used only once (in the traditional method, the PMMA polymer is used for more times, at least three times), thus greatly improving the surface cleanliness of the graphene, retaining the intrinsic high performance of the graphene and being more beneficial to subsequent probe modification.