The ability of Chitosan-Folic acid and PLA-PEG polymers for gene delivery into MCF-7 cells was evaluated. The PLA-PEG copolymer is one of the most appealing nanoparticles for gene transfer to human cells because of its biocompatibility and high blood circulation. However, one of the barriers to the use of these polymers in gene therapy has always been the low effectiveness of these nanoparticles. In this study, Chitosan and folic acid were utilized to improve gene transfer efficiency and encapsulation of PLA-PEG/Chitosan-FA/DNA nanoparticles. PLA-PEG/Chitosan-FA/DNA nanoparticles with different concentrations of Chitosan-FA were prepared using double emulsion-solvent evaporation technique. The biological properties (i.e., biocompatibility, DNA release, and gene transfer ability to MCF-7 cells in vitro) as well as the physical and chemical properties (i.e., particle size, zeta potential, and the morphology of the resultant nanoparticles) of PLA-PEG/Chitosan-FA/DNA nanoparticles were investigated. The results showed that increasing the Chitosan-FA concentration in the PLA-PEG/Chitosan-FA/DNA nanoparticles resulted in an increase in zeta potential, DNA release, encapsulation, and gene delivery efficiency. The MTT assay showed PLA-PEG/Chitosan-FA/DNA nanoparticles exhibit low cytotoxicity and good compatibility. On the other hand, fluorescence microscopy and flow cytometry were used to test the ability of PLA-PEG/Chitosan-FA/DNA nanoparticles with varied concentrations of chitosan-folic acid to transfer the gene to MCF-7 cells. Flow cytometry and fluorescence microscopy analysis showed that increasing the concentration of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles improved gene transfer efficiency to MCF-7 cells.