Iron oxide nanostructured (ION) electrodes were assembled layer-by-layer onto ITO-coated glass substrates and their structure, morphology, and electrochemical properties were investigated, the latter aiming at the development of a chemical sensor for Cu2+. The electrodes were built by immersing the substrate alternately into an aqueous colloidal suspension of positively charged magnetite nanoparticles (np-Fe3O4, 8 nm) and an aqueous solution of anionic sodium sulfonated polystyrene (PSS). The adsorbed amount of both materials was monitored ex-situ by UV-vis spectroscopy and it was found to increase linearly with the number of deposition cycles. The resulting films feature a densely-packed structure of magnetite nanoparticles, as suggested by AFM and Raman spectroscopy, respectively. Cyclic voltammograms of electrodes immersed in acetate buffer (pH 4.6) displayed three electrochemical events that were tentatively ascribed to the reduction of Fe(III) oxy-hydroxide to magnetite, reduction of maghemite to magnetite, and finally oxidation of magnetite to maghemite. The effect of np-Fe3O4/PSS bilayers on the ION electrode performance was to increase the anodic and cathodic currents produced during electrochemical oxidation-reduction of the Fe(CN)(3-/4-) redox couple. With more bilayers, the ION electrode provided higher anodic/cathodic currents. Moreover, the redox couple exhibited a quasi-reversible behavior at the ION electrode as already observed with other working electrode systems. Fitting of voltammetry data provided the apparent electron transfer constants, which were found to be higher in ION electrodes for both redox couples (Fe(CN)(3-/4-) and Cu(2+/0)). By means of differential pulsed anodic stripping voltammetry, the ION electrodes were found to respond linearly to the presence of Cu2+ in aqueous samples in the range between 1.0 and 8.0 x 10(-6) mol x L(-1) and displayed a limit of detection of 0.3 x 10(-8) mol x L(-1). The sensitivity was - 0.6μA/μmol x L(-1). In standard addition and recovery experiments performed with tap water the recovery was about 102%-119%. In similar experiments conducted with ground and instant coffee samples the recovery was 92.5% and 103%, respectively. Furthermore, the ION electrodes were almost insensitive to the presence of common interfering ions, such as Zn2+, Mn2+, Ni2+, and Fe3+, even at concentrations ten times higher than that of Cu2+.