Hydrophilic carbon nanoparticles were utilized as an electroconductive and adsorbent material for membrane-bound [NiFe] hydrogenase obtained from Aquifex aeolicus. Herein, a bottom-up approach was utilized for the preparation of carbon nanoparticulate films on ITO electrodes. The films were prepared either from carbon nanoparticles of opposite charges using a layer-by-layer method, or from negatively charged carbon nanoparticles by their encapsulation in a hydrophobic sol-gel processed silicate matrix. The enzyme was adsorbed on the carbon nanoparticulate film and its catalytic activity towards hydrogen oxidation was studied by cyclic voltammetry and chronoamperometry. The adsorbed hydrogenase exhibits efficient mediatorless electrocatalysis with a maximum activity at temperatures ranging from 60 to 85°C. The effect on the bioelectrocatalytic hydrogen oxidation of adding a mediator to the solution or immobilized on the carbon nanoparticulate film electrodes was also studied.