Novel well-defined amphiphilic fluorinated diblock copolymers P(PEGMA-co-MMA)-b-PC6SMA were synthesized successfully by RAFT polymerization and characterized by FTIR, 1HNMR and GPC. For copolymer coatings, static contact angles, θ, with water (θwater ≥ 109.5°) and n-hexadecane (θhexadecane ≥ 68.9°) pointed to the simultaneous hydrophobic and lipophobic characteristics of the copolymer surfaces. Dynamic contact angle measurements indirectly demonstrated that copolymer films underwent surface reconstruction upon contact with water, which results in a surface with surface coverage of polar PEG units. Moreover, the distinct nanoscale microphase segregation structures were proved by atomic force microscopy (AFM) images. Finally, using bovine serum albumin (BSA–FITC) as the model protein, copolymers exhibited excellent protein adsorption resistance. It is believed that the combination of surface reorganization and nanometer-scale microphase segregation structure endows the excellent protein resistance for amphiphilic fluorinated copolymers. These results provide deeper insight of the effect of surface reconstruction and microphase segregation on the protein adsorption behaviors, and these amphiphilic fluoropolymers can expect to have potential applications as antifouling coatings in the field of marine and biomedical. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41167.