Application of biosensors in non-aqueous media extends their role in biochemical analysis to analytes which cannot be detected by biosensors in water solutions. The behaviour of an enzyme in an organic solvent and the changes expressed in differences of stability, substrate specificity, and sensitivity to inhibitors are discussed. Various methods of biosensor preparation, viz. the use of different electrode systems and ways of enzyme immobilization, are evaluated. A survey of so far designed and used biosensors for non-aqueous media is presented in a table.