Hydrophobic helical hexadecapeptides having an ammonium group were investigated on the formation of an oriented thin layer of the helical peptides on a gold surface. The helical peptides in an ethanol solution were incubated with self-assembled monolayers (SAMs) of carboxylate-terminated alkanethiol on a gold surface to form a helical peptide layer. The thickness of the peptide layer and the orientation of the peptide layer were investigated by the surface plasmon resonance and the FTIR reflection-absorption spectroscopy, respectively. Ion-pair interaction between an ammonium group of the peptides and a carboxylate group of SAMs facilitated the formation of a peptide layer vertically oriented to the substrate. A peptide having an ammonium group and a carboxyl group especially shows a good vertical orientation to the substrate.