Contamination of per- and polyfluoroalkyl substances (PFAS) in water supplies will continue to have serious health and environmental consequences. Despite the importance of monitoring the concentrations of PFAS at potential sites of contamination and at treatment plants, there are few suitable and rapid on-site methods. Many nonconventional techniques do not possess the necessary selectivity and sensitivity to distinguish PFAS from other surface-active components and to quantify the low concentrations in real-world conditions. Herein, we report a novel and rapid method for the detection of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) by leveraging their differential behaviors at the interfaces of emissive complex droplets. Measurement of surface and interfacial tensions via a force tensiometer reveals that PFAS preferentially self-assemble at the water–fluorocarbon oil interface (F/W) rather than the water–hydrocarbon oil interface (H/W). We also observe an opposite behavior for hydrocarbon surfactants. This difference in interfacial behavior produces distinct effects on the morphological change and optical emission of biphasic oil-in-water droplets. The change in the intensity of fluorescence emission, measured with a simple spectroscopic setup, correlates with the concentrations of PFAS. We also demonstrate that the range of detection and sensitivity can be tuned by adjusting the initial composition of the complex droplets. Our results illustrate an alternative mode of sensors that may provide a rapid and on-site detection of PFAS.