The application of nucleic acids as therapeutic agents has been obstructed by the lack of efficient and safe systems for the delivery of the therapeutic DNA into target cells. Nanoparticle (NP) vectors based on biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) polymers, especially the PEGylated ones, seem to be promising drug delivery systems but are far from being ideal gene delivery systems. Given the broad potential of PEGylated PLGA (PEG-PLGA) NPs in the biomedical applications, here, we designed and optimized a PEG-PLGA NP system by incorporating cationic lipids as the excipients for improving the gene delivery efficiency. Systemic investigation of this system by varying the type of the cationic lipids and PEG-PLGA polymers, preparation conditions, transfection conditions and cell lines was carried out, with the aim of finding a relatively efficient formulation for in vivo gene delivery. The optimized NP system showed excellent pDNA transfection efficacy both in vitro and in vivo, while much lower cytotoxicity than commercial Lipofectamine™ 2000 (Lip2000). The prepared system may be very useful for in vivo delivery of gene therapies to targeted cells.