Recognition and detection of melamine are of very significance in food industries. Molecular recognitions of barbituric acid lipids to melamine at the air-water interface have been investigated in detail using in situ infrared reflection absorption spectroscopy (IRRAS). Hydrogen bonding patterns and molecular orientations of the molecular recognitions have been revealed. Prior to molecular recognition, the barbituric acid moieties in the monolayers were hydrogen bonded with a flat-on fashion at the air-water interface, and the alkyl chains were preferentially oriented with their CCC planes perpendicular to the water surface. After molecular recognition, the NH2 stretching bands of recognized melamine were clearly observed at the air-water interface as well as primary characteristic bands, the barbituric acid moieties underwent a change in orientation with non-hydrogen bonded C4=O bonds almost perpendicular to the water surface and C2=O and C6=O bonds involved in hydrogen bonds with melamine, and the alkyl chains were preferentially oriented with their CCC planes parallel to the water surface. The monolayers of barbituric acid lipids exhibited excellent selectivity for melamine over nucleosides.