A mixed (n-octadecyltrichlorosilane (OTS)/[2-(perfluorooctyl)ethyl]trichlorosilane (FOETS)) monolayer was prepared on the water subphase and was subsequently immobilized onto the silicon wafer surface by chemical bonds. Atomic force microscopic (AFM) observation of the mixed (OTS/FOETS) monolayer revealed the formation of a phase-separated structure.In situAFM observation of the adsorption behavior of bovine serum albumin (BSA) onto the mixed (OTS/FOETS) monolayers, successfully showed the adsorption behavior of BSA onto the phase-separated surface. It also revealed that in the case of pH 7.5, BSA was preferentially adsorbed onto the lower surface free energy FOETS phase of the mixed (OTS/FOETS) monolayer. On the other hand, BSA was adsorbed homogeneously onto the OTS and FOETS phases at the isoelectric point of BSA (pI4.7). These results indicate that the preferential adsorption of BSA onto the FOETS phase in the mixed (OTS/FOETS) monolayer system may be due to: (1) the minimization of interfacial free energy between a monolayer surface and an aqueous solution; and (2) the electrostatic repulsion among BSA molecules bearing negative charges.