To improve the circulation time and transfection efficiency of the polyplexes used in gene delivery, a series of Pluronic®/Pluronic®F127–PEI–SS/pDNA complexes (PFPS/pDNA), based on the addition of different kinds of Pluronics® to the reducible disulfide‐bonds‐containing Pluronic®F127–PEI–SS/pDNA (FPS/pDNA) polyplexes, was prepared and evaluated in Bcap and Hela cells in vitro and in vivo. The addition of Pluronics® with molecular weights and hydrophilic–lipophilic balance (HLBs) different from that in the FPS/pDNA complex resulted in five PFPS(1–5)/pDNA complexes, and the correlation between the structure of the free Pluronic® and the properties of the PFPS/pDNA complexes was investigated. The addition of Pluronics® resulted in slightly larger or same‐sized nanoparticles of PFPS/pDNA at a constant N/P ratio. The PFPS copolymer displayed strong stability against DNase I digestion and serum degradation. PFPS‐4 containing added Pluronic® L35, with an intermediate HLB of 19, showed a much higher transfection efficiency and less cytotoxicity than FPS or PEI‐25 kDa in vitro. PFPS‐4 also exhibited a considerably longer blood circulation time than FPS or PEI‐25 kDa in vivo in mice, indicating that the addition of an intermediate Pluronic® can enhance the transfection efficiency of gene delivery systems. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1268–1276, 2014.