The nanohybrid film (NHF) consisted of multi-wall carbon nanotubes (MWTNs) with attached gold nanoparticles was used to immobilize protein. The NHF provides a favorable microenvironment for microperoxidase (MP-11) to perform direct electron transfer (DET) at glassy carbon electrode. The NHF and MP-11 attached covalently were confirmed by transmission electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy. MP-11 immobilized on NHF surface exhibits a surface-controlled quasi-reversible cyclic voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron rate constant (k), formal potential (E-0') and the influence of pH on E-0' in the process were estimated by cyclic voltammetry. The MP-11 immobilized on the NHF retains its bioelectrocatalytic activity to the reduction of H2O2. The apparent Michaelis-Menten constant (K,,) and stability of the H2O2 response were investigated. Moreover, it can catalyze O-2 through four-electron to water in pH 7.0 buffer solution. The kinetic constants for O-2 reduction (k(s) alpha n(alpha)) were obtained. The NHF to immobilize protein with DET shows potential applicability of nanofabrication of biosensors and biofuel cells. (c) 2005 Published by Elsevier B.V.