The invention discloses a preparation method and application of a three-dimensional graphene electrode for an electrochemical biosensor. The preparation method comprises the following steps of: fixing spongy graphene in which industrially produced foam nickel is taken as a substrate and which has a three-dimensional structure and is synthesized through chemical vapor deposition on a glass or quartz sheet; connecting the spongy graphene with the three-dimensional structure and a wire by using a silver conductive adhesive; and coating organic silica gel on a connection point of the metal wire and the graphene for insulation to obtain a spongy graphene electrochemical electrode with the three-dimensional structure. The three-dimensional spongy graphene electrode has the outstanding characteristics of high conductivity, high specific surface area, high electrochemical stability and the like, is easily subjected to surface functional modification, and has high detection sensitivity to dopamine and nicotinamide adenine dinucleotide; and a highly sensitive electrochemical biosensor for non-enzymatically and selectively detecting glucose can be obtained after the surface of the electrode is modified by Co3O4.