A molecularly imprinted self-assembled monolayer (SAM) was fabricated on a gold plate by forming a monolayer with both thiol compound and the template molecule, and removing the template molecules by solvent extraction. 1-Dodecanethiol (DDT), 1-hexadecanethiol (HDT), [1,1':4',1 ''-terphenyl]-4-thiol (TPT), and [1,1':4',1 ''-terphenyl]-4-propanethiol (TPPT) were used as the thiol compounds. Cholesterol was used as the template molecule, and cholesterol, cholic acid, and deoxycholic acid were used as the substrate molecules. Cyclic voltammograms were obtained using these imprinted gold plates as a working electrode, with Ag/AgCl reference electrode and Pt counter-electrode. Potassium ferricyanide was used as a background material for oxidation and reduction. These imprinted monolayers were capable of discriminating cholesterol that had been imprinted. The order of sensing capability of the thiol compounds was TPPT > HDT > TPT > DDT.