In this study, we synthesized quaternary ammonium salt-based gemini surfactants, 2C12(Spacer), with different spacer structures using ethylenediamine derivatives, and investigated their adsorption and aggregation properties by measuring their electrical conductivity, surface tension, fluorescence, and viscosity in conjunction with dynamic light scattering and small-angle X-ray scattering studies to investigate the effect of spacer structures on the properties of the gemini surfactants. The gemini surfactants with spacers containing nitrogen and oxygen atoms were highly soluble in water, whereas those with rigid spacers containing diethylene and triethylene chains exhibited low water solubility. The adsorption and orientation of the gemini surfactants at the air/water interface were significantly affected by the spacer length. Among the synthesized gemini surfactants, the one with the N,N'-dimethylpiperazine spacer showed the highest surface activity. In contrast, the gemini surfactant with the 1-methyl-4-[2-(N,N-dimethylammonio)ethyl]piperazin-1-ium spacer containing an ethylene chain attached to the amino group in the N,N'-dimethylpiperazine spacer (2C12(2/2-N-2)) adsorbed efficiently. However, due to the increased spacer length, this surfactant was unable to orient efficiently at the air/water interface.