Accurately detecting cell secretions in complex microenvironments is crucial for understanding cellular communication, disease progression, and therapeutic responses. Traditional methods, such as ELISA, provide limited insight into the spatiotemporal dynamics of secretions, often requiring invasive or endpoint analyses. To address these challenges, we have functionalized the CellStudio platform—previously developed to integrate cell adhesion areas and microbeads patterns—with a novel self-reporting Structure Switching Signaling Aptamer (SSSA) for Vascular Endothelial Growth Factor (VEGF) detection. This functionalization enables single-step, real-time quantification of VEGF secretion from hundreds of live mesenchymal stem cell (MSC) clusters over a 24-hour period, using standard fluorescence microscopy without the need for cell fixation. The spatial resolution provided by this system enables precise, localized monitoring of secretion events without the need to add extra reagents. This user-friendly platform is easily adaptable to conventional laboratory workflows, offering a versatile tool with potential for studying cellular behavior in real-time with minimal technical barriers.