Here we describe an amperometric assay for enzyme-labeled detection of DNA hybridization based on a redox polymer film, cross-linked and co-immobilized, with a 20-mer oligonucleotide, to pre-adsorbed cysteamine on gold microelectrode surfaces. Hybridization between the immobilized probe DNA and a biotin-modified target DNA (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate and glucose, results in bioelectrocatalytic oxidation of glucose mediated by the redox polymer, generating significant amplification. The use of gold microelectrodes (25 mu m, 40 mu m, 100 mu m diameter), coupled to careful preparation of surfaces, electronic shielding and background subtraction, results in improved analytical performance, compared to that at macroelectrodes, yielding a limit of quantification of ca. 0.6 pM, corresponding to the presence of ca. 2.5 million copies in the 7 mu L assay droplet.