We report a paper-based self-powered sensor patch for prevention and management of exercise-induced hypoglycemia. The article describes the fabrication, in vitro, and in vivo characterization of the sensor for glucose monitoring in human sweat. This wearable, non-invasive, single-use biosensor integrates a vertically stacked, paper-based glucose/oxygen enzymatic fuel cell into a standard Band-Aid adhesive patch. The paper-based device attaches directly to skin, wicks sweat by using capillary forces to a reservoir where chemical energy is converted to electrical energy, and monitors glucose without external power and sophisticated readout instruments. The device utilizes (1) a 3-D paper-based fuel cell configuration, (2) an electrically conducting microfluidic reservoir for a high anode surface area and efficient mass transfer, and (3) a direct electron transfer between glucose oxidase and anodes for enhanced electron discharge properties. The developed sensor shows a high linearity of current at 0.02–1.0 mg/mL glucose centration (R² = 0.989) with a high sensitivity of 1.35 µA/mM.