Layer-by-layer assembly technique was employed to fabricate functional bio-architecture on 3D gold nanorods suitable for working electrode for lactate detection. Successive immobilization of horseradish peroxidase and lactate oxidase on the nanorods was carried out by direct adsorption and specific interaction. The bio-architecture constructed using 3D gold nanorods electrode shows higher sensitivity compared to 20 flat gold electrode due to an increased amount of accessible enzymes and a high transduction capacity. This functional structure system was applied to the determination of L-lactate concentration. The CV results showed a linear response at the 0.1 mM level oft-lactate with a sensitivity of 1.02 mu A/mM. (C) 2012 Elsevier Ltd. All rights reserved.