In this paper, we investigated how different growth conditions (i.e., temperature, growth time, and composition) allows for trading off cost (i.e., In content) and performance of nanostructured indium tin oxide (ITO) for biosensing applications. Next, we compared the behavior of these functionalized nanostructured surfaces obtained in different growth conditions between each other and with a standard thin film as a reference, observing improvements in effective detection area up to two orders of magnitude. This enhanced the biosensor’s sensitivity, with higher detection level, better accuracy and higher reproducibility. Results show that below 150 °C, the growth of ITO over the substrate forms a homogenous layer without any kind of nanostructuration. In contrast, at temperatures higher than 150 °C, a two-phase temperature-dependent growth was observed. We concluded that (i) nanowire length grows exponentially with temperature (activation energy 356 meV) and leads to optimal conditions in terms of both electroactive surface area and sensitivity at around 300 °C, (ii) longer times of growth than 30 min lead to larger active areas and (iii) the In content in a nanostructured film can be reduced by 10%, obtaining performances equivalent to those found in commercial flat-film ITO electrodes. In summary, this work shows how to produce appropriate materials with optimized cost and performances for different applications in biosensing.