A series of well-defined triblock copolymers, poly(N, N-dimethylacrylamide)-block-poly (ethylene oxide)-block-poly(N, N-dimethylacrylamide) (PDMA-b-PEO-b-PDMA) synthesized by atom transfer radical polymerization, were used as physical coatings for protein separation. A comparative study of EOF showed that the triblock copolymer presented good capillary coating ability and EOF efficient suppression. The effects of the M-r of PDMA block in PDMA-b-PEO-b-PDMA triblock copolymer and buffer pH on the separation of basic protein for CE were investigated. Moreover, the influence of the copolymer structure on separation of basic protein was studied by comparing the performance of PDMA-b-PEO-b-PDMA triblock copolymer with PEO-b-PDMA diblock copolymer. Furthermore, the triblock copolymer coating showed higher separation efficiency and better migration time repeatability than fused-silica capillary when used in protein mixture separation and milk powder samples separation, respectively. The results demonstrated that the triblock copolymer coatings would have a wide application in the field of protein separation.