The whole process of octadecyltrichlorosilane (OTS) self assembled monolayer formation on glass surface was clearly revealed by using Atomic Force Microscope (AFM). The growth regularity pattern of OTS self assembled monolayer on glass substrate was obtained by analyses of the AFM images and the average surface mean roughness (%R%-{avg}) of the sample surfaces. The adsorption feature of OTS molecules on the substrate surface during the initial reaction period was probed through a comparison experiment. The whole process of octadecyltrichlorosilane (OTS) self assembled monolayer formation on glass surface was clearly revealed by using Atomic Force Microscope (AFM). The growth regular pattern of OTS self assembled monolayer on glass substrate was obtained by analyzes of the AFM images and the average surface mean roughness (% R % - {avg }) of the sample surfaces. The adsorption feature of OTS molecules on the substrate surface during the initial reaction period was probed through a comparison experiment.