Atomic force microscopy (AFM) is one of the most effective tools in nanotechnology researches. Using the raster scanning method with a soft cantilever, three dimensional sample surface topography can be obtained with a sub-nanometer resolution. In this study, AFM is employed to characterize the nanostructures of polymer film under two temperature treatments. 3-aminopropyltriethoxysilane (APTES) is selected as a model polymer molecule due to its wide applications for amino-functionalization of Si/SiOx based surfaces, such as silicon and mica. The APTES molecule has three ethoxy groups that can react with surface hydroxyl groups or with another APTES molecule through a siloxane bond. The self-assembled APTES self-assembly film has typically three configurations, namely uncrosslinked monolayer, crosslinked monolayer, and multilayer. Ten percent aqueous solution is used to cover the entire mica surfaces. Then the surfaces are rinsed with DI water and dried with N2 to remove left over solution. Two hours incubation was followed in 80°C or 140°C, respectively. At 80 °C (below the boiling point), the uniform APTES film is obtained. On the other hand, at 140 °C (above the boiling point), the film exhibits multilayer structure, as confirmed by AFM images. Contact angle measurement shows that the multilayer film is less wettable than the monolayer film by 20°. It seems that the evaporation rate of the water layer on the substrate is the key to various film configurations.