A biosensor that enables rapid and simple quantitation of a substrate with high accuracy and demonstrates an excellent preservation characteristic by best retaining enzyme activity is disclosed. The biosensor comprises an electrically insulating base plate, an electrode system including at least a working electrode and a counter electrode formed on the base plate, and a reaction layer containing at least an enzyme and a sugar. The reaction layer may be formed on the electrode system. The enzyme may be selected from glucose oxidase, glucose dehydrogenase, and fructose dehydrogenase. The sugar is trehalose, sucrose, glycerol, mannitol, and ribose. If the enzyme selected is glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone, the use of trehalose is preferable.