Abstract Fabrication and organosilane-functionalization and characterization of nanostructured ITO electrodes are reported. Nanostructured ITO electrodes were obtained by electron beam evaporation, and a subsequent annealing treatment was selectively performed to modify their crystalline state. An increase in geometrical surface area in comparison with thin-film electrodes area was observed by atomic force microscopy, implying higher electroactive surface area for nanostructured ITO electrodes and thus higher detection levels. To investigate the increase in detectability, chemical organosilane-functionalization of nanostructured ITO electrodes was performed. The formation of 3-glycidoxypropyltrimethoxysilane (GOPTS) layers was detected by X-ray photoelectron spectroscopy. As an indirect method to confirm the presence of organosilane molecules on the ITO substrates, cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were also carried out. Cyclic voltammograms of functionalized ITO electrodes presented lower reduction-oxidation peak currents compared with non-functionalized ITO electrodes. These results demonstrate the presence of the epoxysilane coating on the ITO surface. EIS showed that organosilane-functionalized electrodes present higher polarization resistance, acting as an electronic barrier for the electron transfer between the conductive solution and the ITO electrode. The results of these electrochemical measurements, together with the significant difference in the X-ray spectra between bare ITO and organosilane-functionalized ITO substrates, may point to a new exploitable oxide-based nanostructured material for biosensing applications. As a first step towards sensing, rapid functionalization of such substrates and their application to electrochemical analysis is tested in this work. Interestingly, oxide-based materials are highly integrable with the silicon chip technology, which would permit the easy adaptation of such sensors into lab-on-a-chip configurations, providing benefits such as reduced size and weight to facilitate on-chip integration, and leading to low-cost mass production of microanalysis systems.