Abstract: As a non viral vector in the gene transfection, of which the transfer process is closely associated with cell membrane, Gemini surfactant has become a major focus of research. In this work, the surface pressure area per molecule (π A) isotherms of the mixed monolayers of Gemini surfactant (18 3 18) and dipalmitoylphosphatidylcholine (DPPC) were measured by Langmuir balance, and then the mean molecular area（Am）, excess area of mixed monolayers (Aex), the excess Gibbs free energy (ΔGex), the Gibbs free energy of mixing (ΔGmix) and the interaction parameter α were quantitatively examined. The mixed nature was determined, and the interaction between 18 3 18 and DPPC was further investigated. In addition, the morphologies of the corresponding LB films were observed by AFM. The results indicate that the stability and themodynamics of mixed monolayer of 18 3 18 and DPPC as well as their interactions were affected significantly by the molar ratio of 18 3 18 and DPPC and the surface pressure. In particular, two minima of ΔGex and ΔGmix for the mixed monolayer of 18 3 18 and DPPC appeared at XDPPC＝0.20 and 0.67, which indicated that the phase separation would occur in the range of 0.20~0.67. This is consistent with the observation by AFM.