Enzymatic biofuel cells (EBFCs) draw much attention since they use renewable fuels to generate electricity and power biological medical devices. However, such EBFCs’ application as devices has always been a puzzle to researchers, let alone their microminiaturization. Herein, we show the efficient embedding of enzymes in a porous reduced graphene oxide (RGO) matrix to form a unique sandwich structure for constructing an on-chip glucose biofuel cell (GBFC). During electrochemical deposition, laccase and glucose oxidase are incorporated in RGO layers to be used as cathode and anode materials, respectively. This GBFC delivers a maximum volumetric power density of 14.77 mW·cm-3 under physiological conditions (50 mM glucose, pH 7). Compared with previous work which is relatively large and cannot be separated from liquid phase system, our on-chip, mediator- and membrane-less micro-GBFC shows great potential to be applied as miniaturized devices to meet the rapidly growing need of being small, thin, wearable and even implantable in modern life.