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Bioelectrocatalysis, which couples enzymatic catalytic and electrode reactions, is the basic principle behind enzyme electrochemical biosensors, biofuel cells, and bioelectrosynthesis systems. Electron mediators facilitate the transfer of electrons between electrodes and oxidoreductases during catalytic reactions, so ideally the mediator remains near the electrode for higher catalytic efficiency. Additionally, electrode materials with three-dimensional space at the enzyme volume mesoscale are expected to improve electrode performance (e.g., increased current density and stability). In previous work, we developed graphene-coated porous silica spheres (G/PSS), which are mesoporous structures filled with conductive carbon microparticles, and then screen-printed G/PSS onto an electrode to form a hierarchical space using the accumulated particles. In this work, we take advantage of this hierarchical space to immobilize a mediator, quinoline-5,8-dione (QD), on a G/PSS electrode. Despite the low potential of QD (-0.075 V vs Ag|AgCl 3 M NaCl, at pH 7.0), it has been shown to be an excellent mediator due to its high reactivity with flavin adenine dinucleotide- or pyrroloquinoline quinone (PQQ)-dependent oxidoreductases. However, applications using QD remain challenging due to its high solubility, which results in fast dissolution of adsorbed QD on a conventional electrode. To solve this issue, we have successfully immobilized the QD to suppress its desorption and dissolution, by simply drying and adsorbing QD onto a G/PSS electrode. This electrode shows significantly improved long-term stability and reusability, while remaining simple to fabricate. Furthermore, we successfully fabricated an enzyme electrode by immobilizing PQQ-dependent glucose dehydrogenase onto a QD-immobilized G/PSS electrode. This method demonstrates that electron transfer does not require free mediator movement and that immobilized QD mediators within pores likely function by allowing localized electron transfer between enzymes and the electrode. The developed technique of immobilizing mediators onto a mesoporous carbon material is expected to be a versatile technique for other enzyme electrode applications.