Electrochemically reduced graphene oxide (ERGO) modified glassy carbon electrode (GCE) surface has been used as a novel immobilization matrix for the oxidoreductase enzyme, catalase (CAT) to explore its direct electrochemistry. In order to attach the CAT molecules firmly to the ERGO modified surface and to enhance the anti-interfering ability of the biosensor a thin layer of nafion (NF) was fabricated above the CAT layer. The as-prepared exfoliated graphene oxide (GO), ERGO, CAT and CAT immobilized ERGO films (NF/CAT/ERGO) were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectra and atomic force microscopy (AFM) studies. The electrochemical behavior and the interfacial changes occurring at all the modified electrodes were probed via electrochemical impedance spectroscopy (EIS) technique. The fabricated NF/CAT/ERGO film based biosensor exhibits quick response (5 s), excellent electrocatalytic activity towards H2O2 in the linear concentration range from 0.05 to 1.91 mM with a sensitivity of 7.76 μA mM–1 cm–2. The proposed CAT biosensor also shows high selectivity towards H2O2, which opens up its practical applications.