The self-assembly nano-structures of type I collagen adsorbed on anionic Gemini surfactant LB monolayer were observed by using atomic force microscopy (AFM) images. It was found that the adsorption behavior and self-assembly structure of collagen could be controlled by the concentration of the collagen solution, adsorption interval and the properties of substrates. With the increase of the adsorption interval and concentration of collagen, the strands size of collagen changed. The self-assembly structures of collagen were also influenced by the interaction between collagen molecules and Gemini surfactant monolayer substrates. Finally, the adsorption behaviors of collagen molecules on cationic Gemini monolayer were compared with those on anionic Gemini monolayer.