A new method to prepare nanopores in a self-assembled monolayer (SAM) of alkanethiol has been developed on the basis of our previous findings that underpotential deposition (upd) of Ag on alkanethiol SAM-coded Au(lll) makes numerous islands of Ag atomic monolayers and upd of Ag or Cu on SAM-coated Au stabilizes the adsorbed alkanethiol molecules against their reductive desorption in an alkaline solution. The procedures are: (i) Ag upd on Au(lll) coated with a decanethiol (DT) SAM to give small islands having DT-SAM/Ag/Au structure, (ii) selective desorption of DT molecules from the Au surface, (iii) Cu upd on the exposed Au surface, (iv) DT-SAM formation on the deposited Cu monolayer, and (v) selective desorption of DT molecules from the surface of the Ag islands. The analysis of the resulting electrode surface with a scanning tunneling microscope revealed that the size and number of the nanopores could be controlled by changing the Ag deposition time in step (i). This method was also available for nanopore preparation in other alkanethiol SAMs by replacing DT with the desired alkanethiol in step (iv). The resulting electrode was found to possess characteristics of a nanoelectrode assembly.