Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-toelectricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O 2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD + , which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O 2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 µW cm-2 , and good operational stability.