Self-assembled lipid films provide new insights into the structure-function relationships of biomolecules at the molecular level. It has potential applications in biology and bionics. In this paper, with regard to atomic force microscopy (AFM) characterization, the surface structures and growth kinetics of self-assembled lipid films as well as their applications in high-resolution AFM imaging of surface-immobilized biomolecules such as proteins, DNA and enzymes are reviewed.