A single nanodroplet of n-decane in a surfactant-free n-decane-in-water emulsion was detected by observingfluorescence emitted from C540 dye molecules dissolved in the droplet on the basis of confocal fluorescencemicroscopy. The fluorescence intensity was found to decrease when a dye-free n-decane-in-water emulsionwas added, because the dye molecules are transferred to the dye-free droplets. Insensitive change of the rateof the dye transfer with the amount of the dye-free emulsion added to the dye-containing one is explained insuch a manner that the permeation of the dye molecules across the droplet−water interface is therate-determining step. The rate-decrease with the diameter of the droplet reveals that the permeability andsolubility of the dye molecules into the droplet tends to increase with the droplet diameter.