Cytochrome c (Cyt c) was successfully immobilized on poly(ferrocenylsilane)-DNA (PFS-DNA) composite film modified gold electrode by electrostatic adsorption. The direct electron transfer of Cyt c in the composite film modified electrode and its application as hydrogen peroxide (H(2)O(2)) biosensor were investigated. The results suggested that Cyt c could be effectively immobilized in the PFS-DNA composite modified electrode. The adsorbed Cyt c showed a good electrochemical activity with a pair of quasi-reversible redox peaks in 0.20 M. pH 7.0 phosphate buffer solution. PFS-DNA composite films showed an obvious promotion for the direct electron transfer between Cyt c and the underlying electrode. The immobilized Cyt c also exhibited a good electro-catalytic activity towards the reduction of H(2)O(2). The catalytic current increased linearly to the H(2)O(2) concentration in a range of 3.0 mu M-1.83 mM (r = 0.999; n = 19) and the detection limit was calculated to be 0.72 mu M based on the criterion of a signal-to-noise ratio of 3. Based on the novel construction, a third-generation biosensor could be obtained for the determination of H(2)O(2). (C) 2010 Elsevier B.V. All rights reserved.