The metal-thiol interface is ubiquitous in nanotechnology and surface chemistry. It is not only used to construct nanocomposites but also plays a decisive role in the properties of these materials. When organothiol molecules bind to the gold surface, there is still controversy over whether sulfhydryl groups can form disulfide bonds and whether these disulfide bonds can remain stable on the gold surface. Here, we investigate the intrinsic properties of sulfhydryl groups on the gold surface at the single-molecule level using a scanning tunneling microscope break junction technique. Our findings indicate that sulfhydryl groups can react with each other to form disulfide bonds on the gold surface, and the electric field can promote the sulfhydryl coupling reaction. In addition to these findings, ultraviolet irradiation is used to effectively regulate the coupling between sulfhydryl groups, leading to the formation and cleavage of disulfide bonds. These results unveil the intrinsic properties of sulfhydryl groups on the gold surface, therefore facilitating the accurate construction of broad nanocomposites with the desired functionalities.