Dicationic (also known as gemini or dimeric) bis-alkylimidazolium surfactants belong to a group of non-viral transfection systems proposed for the successful introduction of different types of nucleic acids (i.e., siRNA, DNA oligomers, and plasmid DNA) into living cells. Our studies reveal the formation of complexes composed of dicationic (gemini) surfactants, 3,3?-[?,?-(dioxaalkane)]bis(1-dodecylimidazolium)chlorides, and 21 base pair deoxyribonucleic acid duplexes (dsDNA). The studied dsDNA and its complex formation process was analysed by small-angle X-ray scattering (SAXS), molecular modelling (dsDNA), agarose gel electrophoresis (AGE) and circular dichroism spectroscopy (CD). We observed the formation of stable complexes for charge ratio values of p/n > 2. Moreover, we noted conformational changes similar to those observed during the transition of B-DNA to C-DNA, X-DNA, and Z-DNA in several spatial structures (i.e., micellar, hexagonal and cubic) formed in mixtures. The surfactants used in this study were investigated for the influence of dioxaalkane spacer length and the presence of an imidazolium moiety on the complexation process. The complexes formed were stable, and the complexation process was reproducible and efficient. Toxicity tests done on HeLa cells allowed for the determination of non-toxic concentrations of studied surfactants. Transfection tests have confirmed that the studied surfactant systems are effective DNA carriers.