The invention belongs to the field of an electrochemical biological fuel cell, and discloses a positive electrode of a glycerin catalytic biological fuel cell and a preparation method and application of the positive electrode. The preparation method comprises the following steps of carrying out surface pre-processing on a substrate electrode, and dropwise adding a Nafion solution to the surface of the electrode to form a layer of Nafion film; inserting the electrode into a Meldola blue aqueous solution for immersion so as to fix the Meldola blue in the Nafion film through ion exchange; taking out the electrode, and washing and drying the electrode to obtain an electrode containing a dielectric layer; and uniformly mixing a graphene-containing chitosan solution, an alcohol dehydrogenase aqueous solution, an acetaldehyde dehydrogenase aqueous solution and a horseradish peroxide aqueous solution according to proportions, dropwise adding the above solutions to the surface of the above electrode, and drying the electrode to obtain the positive electrode of the glycerin catalytic biological fuel cell. The product provided by the invention has the advantages of low cost, a large catalytic and dielectric loading capacity, high catalytic performance and the like, and has favorable application prospect.