In the field of gene delivery, non-viral vectors have become more attractive carriers for nucleic acids since they can overcome the significant drawbacks of viral systems such as safety, immunogenicity, and oncogenicity. Among non-viral vectors, cationic liposome-mediated gene delivery gives promising results for gene therapy approaches. This study aimed to develop cationic liposomes and examine the effectiveness in terms of gene delivery. For this purpose, cholesterol, lecithin, and cationic lipid containing liposomes have been developed by film hydration method. Two different cationic lipids, DDAB and EQ, and their different mole ratios were investigated. Characterization studies showed that obtained liposomes have appropriate physiochemical characteristics (similar to 100 nm, homogenous in size and positive zeta potential) for gene delivery. Gel retardation assay revealed that they have DNA binding and protection ability against nucleases. According to the cytotoxicity evaluation performed on L929 cell line, EQ containing liposomes shows significantly less toxicity comparing DDAB containing liposomes (p<0.05). Furthermore, in vitro transfection study revealed that increasing the EQ mole ratio has increased transfection ability. The stability results showed that the optimal liposome which contains EQ in a higher mole ratio is stable during 90 days at 4 degrees C. Based on these findings, we propose that the developed optimal liposome system in this study could be considered as a suitable nucleic acid delivery vehicle base for gene therapy.