The wide use of portable electronics urgently requires better batteries featuring high energy density, good safety, and biodegradability. Although sugar-powered enzymatic fuel cells (EFCs) could be next-generation, environmentally friendly, micropower sources, they suffer from incomplete oxidation of the sugar fuels and an inability to utilize mixed sugars, which causes the low efficiency of fuel utilization and limits the choice of fuels. In this study, we designed an in vitro 15-enzyme pathway that can co-utilize sucrose, glucose, and fructose in the anodic compartment of EFCs. The EFCs achieved Faraday efficiencies of approximately 95% for these three sugars, suggesting that the fuels were completely oxidized, and yielded a maximum power density of 0.80–1.08 mW cm−2. In addition, EFCs based on this versatile enzymatic pathway were capable of running on mixed sugars, such as commercial soft drinks. These results offer a possible solution for the extraction of the maximum energy stored in mixed sugar fuels and the achievement of high energy densities for EFCs; these EFCs offer good substrate flexibility and commercial potential.