Spontaneous emulsification is a phenomenon that forms nanometer-sized droplets (nanodroplets) without the application of any external force, and the mechanism has been actively studied for application to various technologies. In this study, we analyzed the kinetics of spontaneous emulsification induced by Span 80. The measurement of water concentration in Span 80 hexadecane solution indicated that the chemical potential of water in the nanodroplets decreased as the amount of water in the nanodroplets decreased. Based on this result, water transport between the aqueous phase and nanodroplets in which the chemical potential of water was controlled was quantitatively investigated by using a microfluidic device. The results demonstrate that the kinetics of water transport during spontaneous emulsification induced by Span 80 was described by a model of osmotic transport through an organic liquid film between the aqueous phase and nanodroplets.