In this paper, a series of ABC-type fluorinated amphiphilic triblock copolymer composed of hydrophilic block (PEG block), hydrophobic "anchoring" block (poly(methyl methacrylate) (PMMA) block), and nonpolar hydrophobic block (poly(2-perfluorooctylethyl methacrylate) (PFMA) block) were prepared by atom transfer radical polymerization and characterized. By manipulating the bulk composition and annealing treatment, the films with various surface compositions were prepared and then the adsorption behaviors of human plasma fibrinogen (HFg) and bacterial (Escherichia coli) adhesion on these surfaces were investigated by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The antifouling performances of the amphiphilic triblock copolymers after incorporation of fluorinated block are enhanced compared to PMMA-b-PEG and homopolymer PMMA. The surface compositional heterogeneities with special content of hydrophilic and fluorinated moieties play a significant role in antifouling properties. When the percentage of hydrophobic fluorinated moieties is 21 % and the percentage of hydrophilic ether group is 9.5 % on the surface, the copolymer surface is most unfavorable for protein adsorption. The bacterial adhesion experiment shows the similar results. These results demonstrate the desirable antifouling performance from the ABC-type fluorinated amphiphilic triblock copolymers and provide further realization of the effect of amphiphilic surface structures on the protein adsorption and bacterial adhesion behavior.