A new strategy directed to the durable immobilization of NAD+/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol–gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD+ to the silica matrix using glycidoxypropylsilane in the course of the sol–gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol–gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12 h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD+). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for d-sorbitol, d-glucose and l-lactate with using d-sorbitol dehydrogenase, d-glucose dehydrogenase and l-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.