After showing the failure of conventional gold-enhancement procedures to amplify the gold nanoparticle-based electrochemical transduction of DNA hybridization in polystyrene microwells, a new efficient protocol was developed and evaluated for the sensitive quantification of a 35 base-pair human cytomegalovirus nucleic acid target (tDNA). In this assay, the hybridization of the target adsorbed on the bottom of microwells with an oligonucleotide-modified Au nanoparticle detection probe (pDNA-Au) was monitored by the anodic stripping detection of the chemically oxidized gold label at a screen-printed microband electrode (SPMBE). Thanks to the combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each pDNA-Au, picomolar detection limits of tDNA can be achieved. Further enhancement of the hybridization signal based on the autocatalytic reductive deposition of ionic gold (AuIII) on the surface of the gold nanoparticle labels anchored on the hybrids was first envisaged by incubating the commonly used mixture of AuIII and hydroxylamine (NH2OH). However, due to a considerable nonspecific current response of poor reproducibility it was not possible to significantly improve the analytical performances of the method under these conditions. Complementary transmission electronic microscopy experiments indicated the loss of most of the grown gold labels during the post-enlargement rinsing step. To circumvent this drawback, a polymeric solute containing polyethyleneglycol and sodium chloride was introduced in the growth media to act as aggregating agent during the catalytic process and thus retain the enlarged labels on the bottom of the microwell. This strategy, which led to an efficient increase of the hybridization response, allowed detection of tDNA concentrations as low as 600 aM (i.e., 104 lower than without amplification) and thus offers great promise for ultrasensitive detection of other hybridization events.