Proteins secreted by cells play significant roles in mediating many physiological, developmental, and pathological processes due to their functions in intra/intercellular communication and signaling. Conventional end-point methods are insufficient for understanding the temporal response in cell secretion process, which is often highly dynamic. Furthermore, cellular heterogeneity makes it essential to analyze secretory proteins from single cells. To uncover individual cellular activities and the underlying kinetics, new technologies are needed for real-time analysis of the secretomes of many cells at single-cell resolution. This study reports a high-throughput biosensing microarray platform, which is capable of label-free and real-time secretome monitoring from a large number of living single cells using a biochip integrating ultrasensitive nanoplasmonic substrate and microwell compartments having volumes of ∼0.4 nL. Precise synchronization of image acquisition and microscope stage movement of the developed optical platform enables spectroscopic analysis with high temporal and spectral resolution. In addition, our system allows simultaneous optical imaging of cells to track morphology changes for a comprehensive understanding of cellular behavior. We demonstrated the platform performance by detecting interleukin-2 secretion from hundreds of single lymphoma cells in real-time over many hours. Significantly, the analysis of the secretion kinetics allows us to study cellular response to the stimulations in a statistical way. The new platform is a promising tool for the characterization of single-cell functionalities given its versatility, throughput and label-free configuration.