Nano-emulsion consists of a dispersion of oil droplets sizing below 200 nm, in aqueous continuous phase, and generally stabilized by low-molecular-weight surfactants. These stable nano-carriers are able to encapsulate and transport lipophilic molecules poorly soluble in water. However, the question on the leakage and release mechanisms of an active pharmaceutical ingredient, from oil nano-droplets to an acceptor medium has not been clearly addressed. Herein, we developed a simple fluorescence approach based on self-quenching of lipophilic fluorophore-based on Nile Red (NR668) to monitor cargo transfer from lipid nano-droplets to the acceptor medium. In this method, the fluorophore release can be monitored by the increase in its fluorescence quantum yield and the blue shift in its emission spectrum. The studies of the release process allow emphasizing an important role of the bulk aqueous medium in controlling the droplet to droplet fluorophore transfer and the attained equilibrium. The developed methodology could be applied to monitor release of other lipophilic dyes and it could help to better understand the cargo release from nanocarriers. (C) 2020 Elsevier Inc. All rights reserved.