Using a nonionic. surfactant decaglycerol mono-oleate (MO750), long-term stable ethanol-in-oil (E/O) emulsions were prepared. The Bancroft rule was not applicable to the preparation of E/O emulsion systems. The surface-active performance of MO750 in water differed from that in commercial 95% ethanol and oil. The critical micelle concentration of MO750 could not be measured in 95% ethanol and oil, suggesting that the solvent used greatly affects the surface activity of MO750. The experimental results indicated that MO750 does not significantly reduce the interfacial tension of the 95% ethanol/oil interface, but plays an important role in stabilizing the E/O emulsion. The dispersion behavior of MO750 in 95% ethanol and in oil was investigated with small-angle X-ray scattering. The surfactant was found to exist in a spherical structure with a radius of gyration of about 7 Angstrom in 95% ethanol, and it formed aggregates with a radius of gyration of about 90 Angstrom in oil. A possible stabilization mechanism is presented based on the stratification theory.