Segmented polyurethanes (PUs) containing poly(ethylene glycol) (PEG), poly(propylene glycol), or poly(dimethylsiloxane) soft segments have been prepared by two-step condensation polymerization. Atom force microscopy observation in air and solution indicates that the segmented PU forms it microphase separation oil the surface. By use of quartz crystal microbalance with dissipation and surface plasmon resonance, we have investigated the adsorption of fibrinogen, bovine serum albumin, and lysozyme on it surface constructed by such it PU in aqueous solution in real time. Our results reveal that the protein resistance of the PUs arises from the hydrated PEG segments instead of microphase separation.