In order to construct mio 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.