Arginine plays an important role in cell division and the functioning of the immune system. We describe a novel method by which arginine can be identified using an artificial monolayer based on surface plasmon resonance (SPR). The affinity of arginine binding its recognition molecular was compared to that of lysine. In fabrication of an arginine sensing interface, a calix[4] crown ether monolayer was anchored onto a gold surface and then characterized by Fourier Transform infrared reflection absorption spectroscopy, atomic force microscopy, and cyclic voltammetry. The interaction between arginine and its host compound was investigated by SPR. The calix[4] crown ether was found to assemble as a monolayer on the gold surface. Recognition of calix[4] crown monolayer was assessed by the selective binding of arginine. Modification of the SPR chip with the calix[4] crown monolayer provides a reliable and simple experimental platform for investigation of arginine under aqueous conditions.