The invention belongs to the field of an electrochemical enzyme biological fuel cell, and discloses an enzyme catalysis glycerol oxidized biological fuel cell positive electrode and a preparation method and the application therefor. The preparation method comprises the steps of carrying out surface pretreatment on a base electrode, and uniformly mixing a neutral Nafion solution, a Meldola's blue water solution, and a mixed enzyme solution (ADH to ADLH is equal to 1 to 1) based on a volume ratio of 1 to 1 to 1 to obtain a composite enzyme solution; and adding the composite enzyme solution to the upper surface of the preprocessed electrode in a dropwise manner, and drying the electrode to obtain the enzyme catalysis glycerol oxidized biological fuel cell positive electrode. The produced biological fuel cell positive electrode is low in cost, high in catalyst and medium loading amount, high in catalytic performance and the like, so that the biological fuel cell positive electrode is bright in the application prospect.