The use of cationic polymers in gene therapy aims at improving the circulation, protection and delivery of nucleic acids. The modification of poly(L-lysine) (PLL), including the insertion of hydrophobic moieties, is an efficient strategy to enhance the transfection efficiency of this synthetic delivery system. PLL is usually modified through either the extremities of the polymer chains or the amino groups present on the side chains. Herein the synthesis of dodecalysines as model of PLL with hexyl spacing units regularly distributed along the oligomer backbone is reported. Oligolysines terminated with azidohexyl groups were prepared by solid-phase synthesis and ligated by copper-assisted alkyne-azide cycloaddition using propargylamine. The iterative ligation of α-azidohexyloligolysines was more efficient when performed on resin as compared to the ligation in solution. The plasmid DNA transfection efficiency of the hydrophobically modified dodecalysines was then compared to that of unmodified dodecalysine. The results show that when formulated with the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) the hydrophobized dodecalysines exhibited a higher efficiency than the unmodified one. Interestingly, the efficiency could also be increased by the addition of a fusogenic peptide, which paves the way for modifications of the oligolysines that could lead to improved activities.