The present invention relates to a method of preparing a biofuel cell by using trehalose produced by yeast. Saccharomycetes, Escherichia coli and glucose oxidase are simultaneously immobilized on a carbon felt electrode as an anode, a blank carbon felt electrode is used as a cathode, a saccharomycetes fermentation culture liquid is used as an anolyte, a hydrogen peroxide solution is used as a catholyte, and a proton exchange membrane is disposed between a cathode chamber and an anode chamber to form the biofuel cell. A multiple-enzyme conjugation reaction is designed, has the reaction characteristics of being high-efficiency and precise, and does not use a strong acid, a strong alkali and an organic solvent, environmental pollution can be avoided, and the reaction is green and environmentally-friendly.