Membranekess enzymatic biofuel cells (EBFCs) have the potential to be used in biocompatible devices, such as implantable or wearable devices. However, they face challenges due to low reaction surface area and high resistance attributed to large particle size and protein composition of enzyme catalysts used. In this study, to improve the performance of membraneless EBFCs, three-dimensional graphite felt (3D-GF) electrode was utilized. The 3D-GF electrode has high electrical conductivity and large surface area, allowing the large loading of catalytic components and increasing the reactivity of redox reaction required for EBFC operation. Membraneless EBFC using the 3D-GF electrode is prepared with anodic catalyst including glucose oxidase (GOx) and tetrathiafulvalene mediator, and cathodic catalyst of horseradish peroxidase and GOx. The outermost layer of 3D-GF electrode is coated with gelatin crosslinked by glutar-aldehyde to prevent leaching of components of catalysts. According to evaluations, both anodic and cathodic catalysts are evenly distributed on 3D-GF electrode, and anodic and cathodic currents of 103.5 and 68.7 µA at 0.3 V vs. Ag/AgCl are measured. Additionally, EBFCs using the optimized electrodes demonstrate high power output of 82 µW in a small cell kit size of 1.5×1.5×1.5 cm3, with excellent design flexibility.