In this study, we propose an entirely new combination of materials as a novel formulation devoted for future generation of glucose biosensor. We combined poly(3,4‐ethylenedioxythiophene:polystyrene sulfonate) and poly(vinyl alcohol) as conducting matrix and immobilized the highly active and pioneering enzyme flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) into it. In this context, a good processability of the materials and the preservation of the enzymatic activity are critical steps to provide full device function later on. Thus, we thoroughly characterized the proposed pairing of materials: the influence of the composition on the film‐forming properties was investigated by scanning electron microscopy, optical microscopy, and photography; for the assessment of the enzymatic activity, we developed a spectrophotometric method. A great dependence is observed between activity and composition, ranging from inactive (1.6% retained activity) to highly active (91.5%). A high share of enzyme led to a higher enzymatic activity as long as the necessary condition of a pH higher than 6 was met. These first and fundamental findings can now be used as guidelines to tailor mediator‐free biosensors with enhanced selectivity and to enable the design of devices with improved performance.

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