Nylon-6 nanofibrous membranes (NFM) have been prepared, characterized and used to build-up electrochemical biosensing devices. The assembly and the functioning of biocatalytic NFM are described in connection with the physical and the covalent immobilization of glucose oxidase for the detection of glucose. Effects of the enzyme loading, the mediator, the pH, the surface acidity and the kinetic of the catalysis have been thoroughly investigated. The results show that NFM allow the binding of proteins without the need for the hydrolysis step, in contrast to the nylon film. Furthermore, the high surface-to-volume ratio of the NFM allow superior loading of the enzyme with respect to thin film technology. The immobilization step does not affect the permeability of the coating to the mediator used. These results give evidence that NFM are a promising and inexpensive coating for a novel electrochemical transducer.