The avidin-biotin technology may be used successfully to build stable and spatially ordered assemblies of monomolecular enzyme layers on surfaces as illustrated with the example of glucose oxidase and glassy carbon electrodes. With ferrocene methanol as the cosubstrate, cyclic voltammetry allows a detailed analysis of the catalytic responses, thus providing a demonstration of the spatial order of the multilayer structure and an estimate of the average distance between monomolecular layers. The respective advantages of the avidin-biotin and antigen-antibody technologies are discussed.