The roughness of a multiphase interface and the associated topography between silicone oil and an alcohol-based fluid were measured with an atomic force microscope (AFM) and compared with the results of calculations based upon a capillary-wave model. According to this theory, the interfacial roughness of a liquid liquid interface depends on the density, interfacial tension, and temperature of the liquids. Test samples prepared with both silicone oil and an alcohol-based fluid at various volumetric ratios and controlled temperatures were carefully measured. The experimental results indicate that the interfacial roughness measured with an AFM was consistent with the capillary-wave model. The measured interfacial roughness is influenced mainly by the interfacial tension between the liquids and the temperature-driven Brownian motion of the molecules. Three-dimensional topographical pictures of the interfaces were constructed and archived digitally for subsequent investigation. By employing the outlined method, we examined the microscopic details of interfacial properties, with prospective applications in biochemical and biophysical research.