A biocompatible nanohybrid material (clay/AuCS) based on clay, chitosan and gold nanoparticles was explored. The material could provide a favorable microenvironment for proteins to realize the direct electron transfer on glassy carbon electrodes (GCE). Myoglobin (Mb), as a model protein to investigate the nanohybrid, was immobilized between the clay/AuCS film and another clay layer. Mb in the system exhibited a pair of well-defined and quasi-reversible redox peaks at -0.160 V (vs. saturated Ag/AgCl electrode) in 0.1 M PBS (pH 7.0), corresponding to its heme Fe-III/Fe-II redox couples. UV-vis spectrum suggested that Mb retained its native conformation in the system. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among Mb, AuCS and clay during the modified film drying. Excellent biocatalytic activity of Mb in the modified system was exemplified by the reduction of hydrogen peroxide and nitrite. The linear range of H2O2 determination was from 3.9 x 10(-5) to 3.0 x 10(-3) M with a detection limit of 7.5 mu M based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K-m (Michaelis-Menten constant) were evaluated to be 0.55, 2.66 +/-0.15 s(-1) and 5.10 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.