The search of new genetic vectors that improve the efficacy of gene delivery into diseased cells has led to the creation of several vector formulations that promote effectiveness in applications. However, DNA affinity to vectors as well as vector/DNA complex stability remains a problem to be solved. Here, we study lipoplexes made of DODAB-DOPC cationic liposomes (CL) and calf thymus DNA (ctDNA) as a preliminary cargo gene model of a novel vector formulation to improve DNA delivery efficacy and affinity. Physicochemical characterization was carried out by -potential (), light scattering, ultraviolet, fluorescence spectra, confocal, and electronic microscopy to prove CL and ctDNA complexation and high affinity. Through this approach, and light scattering results indicate an effective CL and ctDNA complexation; CL charge decreases as ctDNA concentration increases. Obtained spectra from UV and fluorescence show high affinity between CL and ctDNA. This can be also verified by confocal microscopy images and electronic microscopy micrographs. All experiments show effective binding between CL and ctDNA, and fluorescence experiments show EtBr displacing as CL concentration increments. CL cytotoxicity assays show high viability when ctDNA is complexed; these results are comparable with those obtained using Lipofectamine 2000. Easy complexation and low cytotoxicity make them a promising formulation for gene delivery. The obtained results from this work motivate us to keep searching for new genetic vector formulations, which could reduce its cytotoxic effect and improve its transfection efficiency, in addition to the low cost of production compared with some commercial products.