The present disclosure provides a three-dimensional hydrogel-graphene-based biosensor and a preparation method thereof, belonging to the technical field of biosensors. The present disclosure provides a three-dimensional hydrogel-graphene-based biosensor, including a substrate, an electrode layer, a graphene film, and a three-dimensional hydrogel material layer that are stacked in sequence; where the three-dimensional hydrogel material layer is formed of a hydrogel material having a three-dimensional network structure; the hydrogel material is obtained by polymerization of raw materials including an acrylamide monomer and a modified probe molecule; and the modified probe molecule is a probe molecule modified with an acrylamide group. The three-dimensional hydrogel-graphene-based biosensor has a desirable stability and a high sensitivity.