Aptamers are single-stranded nucleic acids that fold into unique 3D structures that can mimic antibodies by specific and selective binding to target molecules. For small-molecule targets that have low immunogenicity in particular, aptamers are a valid alternative for developing small-molecule sensing platforms. Despite the myriad of studies on aptamer development, published constructs often fail to progress beyond initial reports, and issues such as false positives have been highlighted. To address this bottleneck, we argue that a deeper understanding of molecular-level events is essential to move aptamer development beyond this cul-de-sac. In this study, we integrate structural, thermodynamic, and electrochemical analyses to investigate the binding and signal-generation mechanisms of DNA aptamers that recognize steroid hormones. It builds on a recently developed pipeline for generating 3D aptamer structures using NMR, exemplified by a testosterone-binding aptamer. The insights gained will enable rational optimization of affinity and selectivity, improve our understanding of dynamics and folding pathways, and be used to inspire new designs for alternative signal-generation mechanisms. Comparing these mechanisms with results from electrochemical assays will aid in formulating guidelines for sensor development. In addition to testosterone, aptamers targeting progesterone and cortisol will be tackled to demonstrate the robustness and broad applicability of our approach.