The invention belongs to the field of electrochemical enzyme biofuel cells, and discloses a biofuel cell anode of enzymatic glycerin and a preparation method and an application of the biofuel cell anode. The preparation method comprises the following steps: carrying out surface pretreatment on a base electrode, dripping a Nafion solution on the surface of the electrode to form a Nafion film; inserting the electrode into a Meldola's blue water solution for soaking, and fixing the Meldola's blue to the Nafion film through ion exchange; taking out the electrode, and cleaning and drying the electrode to obtain the electrode containing a mediator layer; and mixing a chitosan solution containing graphene, a glycerol kinase water solution and a glycerol-3-phosphate oxidase water solution evenly at the ratio, dripping the mixed solution on the surface of the electrode and drying the surface of the electrode to form the biofuel cell anode of the enzymatic glycerin. The product disclosed by the invention has the advantages of low cost, high catalyst and mediator charge capacity, good catalytic performance and the like, and has a good application prospect.