In this study, well-defined amphiphilic cationic triblock copolymers with different lengths of polycationic chain, methoxy poly(ethylene glycol)-b-poly(D,L-lactide)-b-poly(2 dimethylaminoethyl methacrylate) (mPEG-b-PDLLA-b-PDMA), were synthesized and evaluated as carriers for gene delivery. The prepared copolymers can self-assemble into spherical core–shell nanoparticles (NPs). The NPs have a very low critical micelle concentration (CMC) value with only 0.025 mg mL
1. Both copolymers can completely condense the pDNA into spherical complexes when the N/P ratio is equal to or above 3. Bovine serum albumin challenging and DNase I protection assay results demonstrate the mPEG-b-PDLLA-b-PDMA NPs can effectively protect the DNA against protein and nucleases. MTT assay results indicate that mPEG-b-PDLLA-b-PDMA NP/pDNA complexes exhibit obviously lower cytotoxicity compared with commercial gene transfection reagent Lipofectamine 2000/pDNA complexes. Subsequently, in vitro gene transfection studies in HeLa cells without serum show that mPEG113-b-PDLLA10-b-PDMA120 NP/pDNA complexes exhibit higher transfection efficiency than Lipofectamine 2000. Although the transfection efficiency is lower than that in the absence of serum, mPEG113-b-PDLLA10-b-PDMA120 NPs still display equivalent gene transfection efficiency compared to Lipofectamine 2000 when N/P ratio is above 15 in DMEM with 10% serum.