Despite extensive research, nearly all enzymatic fuel cells (EFCs) have failed to achieve continuous discharge, often ceasing within seconds or minutes even in buffered media─revealing a deep unresolved bottleneck in enzyme–electrode coupling. Here, we identify and experimentally validate the underlying mechanism behind this limitation, showing that excessive immobilization suppresses enzyme dynamics, while free dispersion decouples electron transport─together defining the core confinement–activity trade-off that has constrained EFC performance for decades. To overcome this, we introduce a bilayered rolled-up titanium–enzyme nanomembrane (BRUTENE) architecture that enables dense enzyme confinement with preserved catalytic mobility and rapid electron evacuation. Operating in both phosphate-buffered saline (PBS) and undiluted human serum, BRUTENE-based EFCs achieve near-theoretical power density and sustain continuous discharge for over 175 h─representing a multiorder-of-magnitude improvement over all previously reported systems and experimentally validating the proposed structural mechanism. Moreover, using Faraday’s law of electrolysis, we quantitatively verify for the first time the electron transfer stoichiometry of glucose oxidation: two electrons from primary oxidation and two from a previously unconfirmed secondary oxidation step in serum, yielding a total four-electron pathway. This dual discovery of both the structural origin of EFC instability and the mechanistic validation of glucose multielectron catalysis establishes a new framework for high-performance, physiologically compatible bioenergy systems.