A new layer-by-layer supramolecular approach for the construction of self-assembled nanoarchitectures of polyamidoamine (PAMAM) dendrimers and peroxidase on gold surface is reported. The methodology is based on the supramolecular self-assembly of alternated layers of adamantane-modified horseradish peroxidase and beta-cyclodextrin-branched PAMAM G-5 dendrimers on a gold electrode, previously coated with beta-cyclodextrin-modified cysteamine core PAMAM G-4 dendron. The formation of layer-by-layer assemblies (up to three dendrimer/peroxidase bilayers) was studied by SPR, quartz crystal microbalance, AFM and cyclic voltammetry. The analytical applicability of these architectures was evaluated by constructing a H2O2 biosensor. The electroanalytical response of the biosensor towards H2O2 increased with the number of enzyme layers. The bioelectrode constructed with three enzyme layers showed a low detection limit of 160 nM, a sensitivity of 602 mu A/M cm(2) and retained 63% of its initial activity after 30 days of storage in wet conditions. (C) 2012 Elsevier Ltd. All rights reserved.