Methacryloyl-terminated poly[methyl(3,3,3-trifluoropropyl)siloxane] (PMTFPS-MA) macro-monomers with controlled segment length were designed and synthesized by anionic ring-opening polymerization of 1,3,5-trimethyl-1,3,5-tri(3',3',3'-trifluoropropyl)cyclotrisiloxane (F-3). Then, a series of novel amphiphilic fluorosiloxane-containing graft copolymers were prepared using PMTFPS-MA, poly(ethylene glycol) methyl ether methacrylate (PEG-MA) and methyl methacrylate (MMA) as the co-monomers, and their surface properties and protein-resistant performance were investigated. It was found that the composition of amphiphilic graft copolymers would play an important role in the protein-resistant properties. When the copolymers contain 65.5 wt% PEO segments and 6.4 wt% PMTFPS segments, the coating surface exhibits strong protein-resistant property and shows almost no protein adsorption on it. Based on the surface analysis of amphiphilic graft copolymer films both in dry and wet conditions, the protein-resistant property of amphiphilic copolymers can be owing to the transition from a hydrophobic surface to hydrophilic surface with highly efficient water-driven PEO surface migration. The PEO segments are utilized to prevent protein adsorption whereas the low surface energy PMTFPS segments are utilized to drive away the adsorbed proteins.