Nanostructured indium tin oxide (ITO) surfaces present an interesting yet unusual combination of properties (high electrical conductivity and optical transparency) at a high surface-to-volume ratio. Thus, previous studies presented nanostructured ITO electrodes as potentially suitable platforms for electrochemical biosensors, but still there is a lack of research on the optimization of preparation methods for such electrodes. We present a systematic study on the properties of nanostructured ITO electrodes prepared by physical deposition, where the substrate temperature was tuned for achieving the best combination of structural properties (namely electrical conductivity and optical transparency) and electrochemical performance. Analysis of faradaic cyclic voltammetry (CV) was performed to determine the electroactive surface area of the samples, and these results were benchmarked against those obtained by non-faradaic CV and Mott–Schottky (MS) analysis. The latter was useful to determine the dependence of some intrinsic features of the semiconductor on the substrate temperature during deposition. The results show that, out of a wide temperature range covering from 200 °C to 500 °C, there is a two-phase temperature-dependent growth, explained by the Stranski–Krastanov and self-catalytic vapor–liquid–solid (VLS) methods, and, on the other hand, that there is an optimal growth temperature at 300 °C that maximizes the electroactive surface area and sensitivity. This means that cost-effective electrodes can be prepared at low temperatures outperforming in terms of electroactive surface area, surface capacitance and sensitivity. As a proof-of-concept, nanostructured ITO electrodes were electrochemically derivatized with aryl diazonium salts (as a first step towards biochemical functionalization), and the performance of the optimized electrodes was tested in a real scenario.