We demonstrate the feasibility of optical biosensing using a material which, in essence, is a modified inorganic film to which various enzymes were covalently attached. Thin and transparent blue films composed of Prussian blue and incorporated into a network of N-substituted polypyrroles are sensitive to pH in the 5–9 range at 720 nm wavelength and can be modified with enzymes to result in the respective biosensors. Several methods of enzyme immobilization, using bifunctional crosslinking reagents, and various enzymes were tested. The best results were obtained using the one-step carbodiimide method which resulted in highly active, stable and transparent biosensor films for optical determination of urea and acetylcholine. The operational stability exceeded 1 month and even after 2 months of dry storage at room temperature the activity did not drop. The biosensors allow optical determination of the respective substrates in the millimolar concentration range.