Polyethylene glycol or phosphorylcholine is often introduced into polycationic non-viral vectors to inhibit the non-specific protein adsorption. However the ability of vectors to condense DNA and the cellular internalization of complexes are unavoidably compromised. In this work, a polysulfobetaine-cationic methacrylate copolymer: 2-(dimethylamino) ethyl methacrylate-block-(N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide) (PDMAEMA-b-PMPDSAH) diblock copolymer was synthesized via atomic transfer radical polymerization method and investigated as a new non-viral vector for gene delivery. Incorporation of polysulfobetaine into cationic methacrylate retained a better DNA condensation capability. MTT assays revealed that the cytotoxicity of PDMAEMA 200-PMPDSAH n copolymer was lower than that of PDMAEMA 200. PDMAEMA 200-PMPDSAH 80 which was much superior to its homopolymer in mediating gene transfection demonstrated comparable efficiency to PEI25 kDa at a weight ratio of 8 in the presence of 10% serum. At higher serum contents, the transfection of PDMAEMA 200 and PEI25 kDa was deteriorated, whereas PDMAEMA 200-PMPDSAH 80 still retained better transfection efficiency, 4–5 fold more effective than PEI25 kDa. For the sake of comparative study, we synthesized structurally similar copolymer from DMAEMA and 2-methacryloyloxyethyl phosphorylcholine, PDMAEMA 200-PMPC 80. PDMAEMA 200-PMPDSAH 80 exhibited much higher gene transfer levels than PDMAEMA 200-PMPC 80 under the same conditions. The results of flow cytometry indicated that highly hydrophilic MPC block profoundly impeded the cellular internalization of nanocomplexes; in contrast, incorporation of polysulfobetaine remained the increased cellular uptake. Differential scanning calorimetry assay of thermodynamic phase transition of dipalmitoyl- sn-glycero-3-phosphocholine(DPPC) induced by polymer vectors demonstrated that MPC only marginally contributed to the perturbation of DPPC; polysulfobetaine facilitated more evident perturbation of DPPC bilayer instead, an indication that polysulfobetaine units could aid in the endocytosis of nanocomplexes.