Hydrophobic surfaces in contact with aqueous media are omnipresent in nature. A plethora of key biological and physiological processes occur at the interface of immiscible fluids. Besides its fundamental importance, probing such interfaces is rather challenging, especially when one medium is bathed in the other. Herein, we demonstrate a fluorescence-based method that probes the oil–water interface and interfacial processes through surface dielectric perturbations. The fluorescence response of Nile Red is measured in hexadecane in water nanoemulsions. Three major spectral components appear: two from the bulk liquid media (hexadecane and water) and a distinct band at around 640 nm due to the interfacial component. Such spectra are deconvoluted using the multivariate-curve-resolution algorithm, and interface-correlated fluorescence spectra are attained. The influence of anionic sodium dodecylbenzenesulfonate (SDBS) and cationic cetyltrimethylammonium bromide (CTAB) surfactants on the oil–water interface is elucidated with concentration-dependent measurements. A charge-dependent spectral shift is observed. The interface correlated band at 641 nm for bare hexadecane nanoemulsions red shifts in the presence of anionic surfactants, indicating an apparent dielectric increase. In contrast, the same band gradually blue shifts with increasing cationic surfactant concentration, indicating an apparent interface dielectric decrease. Such a method can be utilized to probe alterations at interfaces beyond the oil/water interface.