Using zinc powders as source material, ZnO nanorods (ZnONR) were fabricated on gold wire by a hydrothermal reaction without any other surfactant. The gold wire end was coated by a thin layer of Zn-Au alloy to improve the nucleation for growth of ZnO nanostructures and to further improve the performance of the biosensor, which was constructed by alternatively immobilizing poly(sodium 4-styrenesulfonate) (PSS) and horseradish peroxidase (HRP) on the ZnONR. Electrochemical measurement, ultraviolet-visible spectrum, zeta-potential, and scanning electron microscopic analysis demonstrated that PSS and HRP were stably adsorbed layer by layer on the ZnONR surface, and the HRP kept bioactivity for H2O2 detection without an electron transfer mediator. The multilayered HRP sensors exhibited a wide linear range and low detection limit. The sensitivity of the biosensor increased with the immobilized HRP layers from the lowest value of 36.28 mu A mM(-1) for a monolayer.