In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilus is a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono-and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.