Escherichia coli's copper efflux oxidase (CueO) has rarely been employed in the cathodic compartment of enzymatic biofuel cells (EBFCs) due to its low redox potential (0.36 V vs. Ag/AgCl, pH 5.5) towards O reduction. Herein, directed evolution of CueO towards a more positive onset potential was performed in an electrochemical screening system. An improved CueO variant (D439T/L502K) was obtained with a significantly increased onset potential (0.54 V), comparable to that of high-redox-potential fungal laccases. Upon coupling with an anodic compartment, the EBFC exhibited an open-circuit voltage (V ) of 0.56 V. Directed enzyme evolution by tailoring enzymes to application conditions in EBFCs has been validated and might, in combination with molecular understanding, enable future breakthroughs in EBFC performance.