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Nonviral lipid-based vectors are promising transporting systems for the intracellular delivery of therapeutic gene sequences and directly influence the success of gene delivery. However, the associated drawbacks like lower transfection, toxicity, and targetability require further improvement. Thus, herein, we report a novel lipid formulation by the mixing of two distinct cationic surfactants such as tocopheryl succinate based cationic lipid and 1,12 dodecane based bolaamphiphile and prove it to be a good transfection reagent with its competing potential with the "golden standard", Lipofectamine 3000 (L3K). These interesting aggregations were named "Bolaliposome" and showed adequate unilamellar vesicle morphology under transmission electron microscopy, having a size of around 100 nm and could transfect efficiently different varieties of cell lines. Moreover, the generated complexes from bolaliposome and DNA (bolalipoplex) were characterized in terms of surface potential, hydrodynamic size, and gel electrophoresis. Various pharmacological inhibitors were also used in reporter gene expression to prove that the complexes followed the clathrin-mediated endocytosis. Finally, these findings would be helpful in the making of new aggregates and the development of better cytofectins. This was developed by optimizing the formulation based on the efficiency of reporter gene expression performed using the pEGFP-N3 plasmid.