An innovative nanoplasmonic biosensor was presented for real-time label-free monitoring of cytokine secretion at single-cell resolution. This biosensor is integrated with a uniquely designed microfluidic device that provides a microchamber for cell incubation and fluid regulation for stable live cell monitoring. Different spatiotemporal profiles of interleukin-2 secretion were observed from single lymphoma cells. This novel configuration of label-free biosensor is promising for single-cell related studies.