A new sensor design is reported for the construction of an amperometric enzyme biosensor toward H2O2. It was based in the supramolecular immobilization of alternating layers of horseradish peroxidase (either modified with 1-adamantane or beta-cyclodextrin-branched carboxymethylcellulose residues) on An electrodes coated with polythiolated beta-cyclodextrin polymer. The analytical response of the electrodes, using 1 mM hydroquinone as an electrochemical mediator, increases when the number of enzyme layers increases. The biosensor having three enzyme layers showed a sensitivity of 720 mu A/M cm(2) and a detection limit of 2 mu M and retained 96% of its initial activity after 30 days of storage. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.