In the present study, we investigated the surface reorganization behaviors and adsorption conformations of fibrinogen on the surface of polyrotaxanes containing different amounts of α-cyclodextrin (α-CD) by using surface-sensitive vibrational spectroscopy sum frequency generation (SFG). For comparison, behaviors of the surface restructuring and fibrinogen adsorption on the random copolymers containing similar terminal groups were also investigated. It was found that larger amounts of BMA moieties of polyrotaxanes form ordered surface structures after immersion in water for 48 h. Furthermore, the polyrotaxane surfaces exhibit a much higher capability of fibrinogen adsorption than the random copolymer surfaces. The water-induced surface restructuring of the polyrotaxane films slightly affects the adsorption structure of the fibrinogen molecules.