In order to construct two dimensional DNA-mimetics, nucleo base monolayers, composed of amphiphilic adenine and thymine derivatives, were prepared at the air–water interface. The monolayer were characterized by pressure–area isotherm measurements, UV-Vis absorption and FT-IR reflection-absorption spectroscopy (RAS), and fluorescence spectroscopy and microscopy. A 1:1 mixture of octadecyladenine (C 18-Ade) and octadecylthymine (C 18-Thy) formed the most condensed monolayer on a pure water subphase in the three combinations of the equimolar mixtures of C 18-Ade, C 18-Thy, and octadecylcytosine (C 18-Cyt). A FT-IR RAS spectrum of the transferred monolayer suggests the base-pairing with Watson–Crick type hydrogen bonds in the C 18-Ade/C 18-Thy monolayer. An in situ observation of fluorescence image and spectrum at the air–water interface indicates that the Watson–Crick type monolayer can incorporate an amphiphilic intercalator, octadecylacridineorange (C 18-AO), as well as DNA.