Highly organized (3-mercaptopropyl)trimethoxysilane (3-MPT) films have been prepared via self-assembled coupled with sol-gel linking technology. Horseradish peroxidase (HRP) is successfully immobilized onto the densely packed three-dimensional (3D) 3-MPT network and the direct electrochemistry of HRP is achieved without any electron mediators or promoters. Redox thermodynamics of HRP on the 3-MPT films, which is obtained from the temperature dependence of the reduction potential, Suggests that the positive shift of redox potentials of HRP at the interface of 3-MPT originates from the solvent reorganization effects and conformational change of the polypeptide chain of HRP. Based on the direct electrochemistry and electrocaralytic ability of HRP, a sensitive third-generation amperometric H2O2 biosensor is developed with two linear dependence ranges of 5.0 x 10(-7) to 1.0 X 10(-4) and 1.0 x 10(-4) to 2.0 x 10(-2) mol L-1. (C) 2008 Elsevier Ltd. All rights reserved.