Abstract: The influence of equilibrium time, concentration of salt and spacer length on the interaction between the cationic Gemini surfactant(［C18H37(CH3)2N+ (CH2)s N+(CH3)2C18H37］·2Br-, abbreviated as 18 s 18,s=3、4、6、8、10、12) and DNA on the air water interface were investigated using LB film and AFM. We found that for the interaction between the Gemini surfactant and DNA it takes a long time to achieve balance. The interaction between Gemini surfactant and DNA declined with the increase of salt concentration, because the addition of salt shielded the surface charge of DNA molecules. When spacer is rigid (s=3 and 4), the intermolecular steric hindrance is relatively small, and the main morphology of Gemini/DNA complex film is parallel fibril .When spacer is flexible (s≥8), the intermolecular steric hindrance increased with the increasing of spacer length, and the main morphology becomes sheet aggregates.