In this study, the interface characteristics during the two-phase invasion of silicon oil and water were investigated using high-precision optical experiments and molecular dynamics simulations. An experimental system composed of a CCD (charge coupled device) industrial camera and an optical microscope was designed to observe the interface state between the dispersed phase at the silicone oil-water interface. At the same time, the microscopic image and energy spectrum results of the silicone oil-water mixture were obtained using a cryo-scanning electron microscope (Cryo-SEM). The molecular dynamics method was also used to simulate the dynamic process of the mass transfer of the silicone oil-water mixture. Through experimental results and molecular dynamics simulations, the phenomenon of diffuse mass transfer during the phase invasion and the existence of silicon oil-water associations were confirmed. The results showed that the silicone oil and water molecules were associated with each other due to the hydrogen bonding and van der Waals interactions, but the interfacial association matter was loose and unstable. The C–H, Si–O, and H–O bonds were the main factor in the formation of the silicone oil-water dispersion association matter. The findings revealed the rationality of the dispersion associated substances at the silicon oil-water interface, and showed its microstructure characteristics. The study is expected to provide an insight for the study of silicon oil-water interface and enrich the emulsification and separation theories between silicon oil and water solutions.