Real time assay of protein secretion from living single cells was realized by coupling total internal reflection fluorescence (TIRF) microscopy observation and an optically optimized microwell array device. This system was based on a sandwich immunosorbent assay. Individual cells were cultured on an inverted microscope in microwell arrays fabricated of an amorphous fluoropolymer, CYTOP, to be monitored by TIRF imaging. CYTOP have no interference for the TIRF imaging because the refractive index of CYTOP is almost same as that of water. Using this system, we could succeed in real time secretion assay of lipopolysaccharide stimulated mast cells and the secretion time course of IL-1β from a single MC/9 cell was obtained successfully.