Trehalose, a disaccharide formed by an,-1,1-glucosidic bond between two -glucose units, is the principal sugar circulating in the blood or hemolymph of most insects. Insect cyborgs have been developed for utilizing search and rescue robots in disasters and as environmental monitoring robots. This study aimed to develop the anode for high-powered compact enzymatic fuel cells utilizing trehalose to power electronic devices implanted in insects. Single-walled carbon nanotubes (SWCNTs) and gold nanoparticles (AuNPs) were sequentially adsorbed on the surface of carbon paper. Glucose oxidase (GOx) was adsorbed on the electrode surface, whereas two enzymes, glucose oxidase and trehalase (TREH) were separately immobilized via gelatin on the electrode. Electrochemical analyses were performed using cyclic voltammetry and electrochemical impedance spectroscopy. Analyses using TEM, SEM, and SEM-EDX showed that SWCNTs and AuNPs were well distributed on the surface of the electrode. The introduction of SWCNTs and AuNPs improved electrode performance by reducing the electron transfer resistance of the electrode. The highest current was generated by carbon paper modified with SWCNTs and AuNPs when GOx was immobilized. Bi-enzyme anode was fabricated based on carbon paper modified with SWCNTs and AuNPs. The cathode was also fabricated by immobilizing bilirubin oxidase (BOD) on this nanostructured electrode. Biofuel cell was assembled and was tested using trehalose as fuel. The highest power density, 32.3 µW/cm2, was obtained at 86.7 µA/cm2 of discharging current density.