The invention discloses a biosensor of a graphene/copper particle mixing structure and a preparation method thereof. According to the biosensor, a layered structure formed by mixing two-dimensional graphene and copper nanoparticles is attached to the upper surface of glass fiber paper. The preparation method comprises the following steps: coating a copper film on the glass fiber paper by adopting a vacuum coating method, depositing the produced graphene on the copper film by adopting chemical vapor deposition, and treating the copper film into a copper nanoparticle layer, so that the copper film forms the layered structure formed by mixing the two-dimensional graphene and copper nanoparticles.