H2O2 is a product of reactions catalysed by several oxidase enzymes and it is essential in environmental and pharmaceutical analyses. The most commonly used enzyme in understanding the biological behaviour of catalysed oxidation of H2O2 is horseradish peroxidase (HRP). In our experiments HRP was bound to carboxyl-functionalized multiwalled carbon nanotubes (MWNT-COOH) by N-hydroxysuccinimide (NHS) and 1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide (EDC) crosslinkers. The activity of this bio-nanocomposite and the limit of detection (LOD) for H2O2 were determined by measuring the fluorescence of tetraguaiacol (which chemical is the product of guaiacol oxidation after addition of H2O2 to the reaction mixture) as a function of time. The hydrogen peroxide biosensor we developed exhibited a detection limit of 1.2 µM H2O2 s−1 which resolution was better than the one measured in solution by about a factor of eight (it was 10 µM H2O2 s−1 in solution). An attempt has been made to measure the concentration of H2O2 in an electrochemical cell with HRP immobilized on the surface of an electrode made of indium tin oxide (ITO, a transparent conductive oxide) and MWNT.