After initial testing and optimization of anode biocatalysts, a membraneless glucose​/oxygen enzymic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DHCDH) were tested during the pre-​screening. The enzymes were mixed, wired and entrapped in a low-​potential Os-​complex-​modified redox-​polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the c.d. for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-​glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDHCtCDHC310Y) with a high glucose-​turnover rate in combination with an Os-​complex-​modified redox polymer with a high concn. of Os complexes as well as a low-​d. graphite electrode. The optimized biofuel cell with the AmPDH​/ngDHCtCDHC310Y anode showed not only a similar max. voltage as with the biofuel cell based only on the ngDHCtCDHC310Y anode (0.55 V) but also a substantially improved max. power output (20 μW​/cm2) at 300 mV cell voltage in air-​satd. physiol. buffer. Most importantly, the estd. half-​life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.