Nanosized polymer-enzyme films were fabricated on graphite surfaces via a sequential two-stage adsorption of the pH- and thermoresponsive polymer, viz., linear poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), and the enzyme, viz., choline oxidase (ChO). The influence of the conditions (pH and temperature), which are applied for deposition of the polymer onto graphite surface, on electrochemical responses of the polymer-enzyme sensor constructs and kinetic parameters of enzymatic oxidation of choline by ChO integrated into the polymer-enzyme films was revealed. Stimuli-sensitivity of PDMAEMA was found to control the properties of the formed polymer coatings (surface coverage, film thickness and roughness), which decisively determine the amount of the ChO bound by the polymer film and, consequently, the resultant electrochemical characteristics of the polymer-enzyme sensor constructs.