The hydrophobic interfacial characterization of sugar–ester alcohol W/O microemulsion was investigated in order to develop a suitable design for reaction media. A W/O microemulsion system was prepared as a combination of sugar–ester, DK-ester-F-110 with alcohol as a cosolvent. The diameter of the sub-micron-sized water pool, the hydrophobicity of the water pool, and the fluidity of the micro-interface were studied to analyze the reactivity of Rhizopus delemar lipase in lipid hydrolysis. The diameter of the sub-micron sized water pool was measured by dynamic laser light scattering. It was proportional to the 0.33 power of W soln and decayed with increasing alcohol concentration. The initial reaction rate of the hydrolysis of triolein in W/O microemulsion depended on the solubilized water content. The hydrophilic character of the micro-water pool was estimated from the fluorescence wavelength indicated by Coumarin 343. The maximum initial reaction rate appeared at 450–460 nm. The fluidity of the micro-interface was investigated by fluorescence anisotropy using TMA-DPH. A suitable physicochemical reaction condition for high reactivity to occur was found, satisfying both the hydrophilicity and the fluidity of the micro-interface.