It is a well-known fact that various cytokines and interferons secreted from effector cells play a crucial role in achieving various complicated functions in a living body. On the other hand, the effector cells secreting the humoral factors are often assumed to be only a small part of the population. Therefore, techniques to distinguish secretion activity at the single cell level, e.g. intracellular FACS and Elispot assay, are widely used. Recent single cell studies have revealed that secreted cytokines from cells are heterogeneous in terms of amount and variety even when the same stimulus for secretion response is given to the cells considered to be identical. These facts concerning secretion heterogeneity also highlighted the new issues of how to overcome heterogeneity to maintain the robustness of the organism and how to make clinically meaningful measurements from fluctuating features. Here we propose a new technology to continuously monitor cytokine secretion activity of individual cells over a wide period of time, “live cell imaging of secretion (LCI-S)”. This method is based on our previously reported technology (Sci. Rep., 4, 4736, 2014; Cell Rep., 8, 974-982, 2014), where we could continuously monitor sandwich fluoroimmunoassay on total internal reflection fluorescence illumination microscopy without any washing steps. We improved LCI-S for long-term continuous measurement of secretion activities by realizing the essential perfomance for clinical evaluation, i.e. long-term stability of the measurement for several days or more, simultaneous multi-specimen or multi-condition measurement and multi-cytokines measurement. Furthermore, we successfully recovered each cell exhibiting characteristic secretion activity and analyzed gene expression by single cell RNA-seq. We applied our LCI-S to several type of cytokine secretion response including inflammasome assosicated IL-1b production of macrophage and type 2 cytokine production of type 2 innate lymphoid cells.