Zirconia/multi-walled carbon nanotube (ZrO(2)/MWCNT) nanocomposite was prepared by hydrothermal treatment of MWCNTs in ZrOCl(2)center dot 8H(2)O aqueous solution. The morphology and structure of the synthesized ZrO(2)/MWCNT nanocomposite were characterized by transmission electron microscopy and X-ray diffraction analysis. It was found that ZrO(2) nanoparticles homogeneously distributed on the sidewall of MWCNTs. Myoglobin (Mb), as a model protein to investigate the nanocomposite, was immobilized on ZrO(2)/MWCNT nanocomposite. Ultraviolet-visible spectroscopy and electrochemical measurements showed that the nanocomposite could retain the bioactivity of the immobilized Mb to a large extent. The Mb immobilized in the composite showed excellent direct electrochemistry and electrocatalytic activity to the reduction of hydrogen peroxide (H(2)O(2)). The linear response range of the biosensor to H(2)O(2) concentration was from 1.0 to 116.0 mu M with the limit of detection of 0.53 mu M (S/N = 3). The ZrO(2)/MWCNT nanocomposite provided a good biocompatible matrix for protein immobilization and biosensors preparation. (C) 2010 Elsevier Ltd. All rights reserved.