Vertically aligned nanowire array electrodes (NAEs) were prepared by electrodeposition of gold into an anodic aluminium oxide membrane (AAM), providing an ordered three-dimensional (3D) matrix for immobilization of redox proteins. Third-generation H2O2 biosensors were prepared by covalent immobilization of horseradish peroxidase (HRP) on the self-assembled monolayer modified NAEs. Direct electron transfer and electrocatalytic performances of the HRP/NAEs with different nanowire lengths (deposition time of 2, 4 and 5 h) were investigated. Results showed that with longer nanowires, better performances were achieved. The HRP/NAE(5h) (5 h deposition time) exhibited remarkable sensitivity (45.86 mu A mM(-1) cm(-2)) towards H2O2 with a detection limit of 0.42 mu M (S/N=3), linearity up to 15 mM and a response time of 4 s. The ordered 3D gold nanowire array with high conductivity, excellent electron transfer capability and good biocompatibility proved promising for fabricating sensitive, selective, stable and mediator-free enzymatic biosensors. (C) 2009 Elsevier B.V. All rights reserved.