A novel nanohybrid material, constructed by gold nanoparticles (GNPs) and multiwalled carbon nanotubes (MWNTs), was designed for immobilization and biosensing of myoglobin (Mb). Morphology of the nanohybrid film was characterized by SEM. UV-vis spectroscopy demonstrated that Mb on the composite film could retain its native structure. Direct electrochemistry of Mb immobilized on the GNPs/MWNTs film was investigated. The immobilized Mb showed a couple of quasireversible and well-defined cyclic voltammetry peaks with a formal potential of about À 0.35 V (vs. Ag/AgCl) in pH 6.0 phosphate buffer solution (PBS) solution. Furthermore, the modified electrode also displayed good sensitivity, wide linear range and long-term stability to the detection of hydrogen peroxide. The experiment results demonstrated that the hybrid matrix provided a biocompatible microenvironment for protein and supplied a necessary pathway for its direct electron transfer.