Water is arguably the most common and yet least understood material on Earth. The interface between water and a hydrophobic medium, such as air, oil, or lipids, plays a fundamental role in chemistry and biology. However, the behavior of molecules at interface of micron-sized water droplets (microdroplets) in such media is poorly characterized. Herein we employed two-photon fluorescence microscopy and Forster resonant energy transfer imaging to study the probe localization in water-oil microdroplets with high contrast and resolution. We found that there exists a general effect of surface enrichment and orientation alignment for water-soluble probes. Remarkably, probes are concentrated into a similar to 10 nm thin layer at the microdroplet water-oil interface by up to 10 000-fold compared to the bulk counterpart. We suggest that the strong enrichment and alignment of water-soluble molecules, likely to be induced in part by a local electric field at the interface, could be a major factor accounting for orders of magnitude faster reaction rates observed in aqueous microdroplets compared to their bulk counterparts.