The surface pressure-area isotherms of bilirubin (H2B R) monolayers at an air-water interface on subphases with different pH values and on subphases containing metal ions such as Ca2+, Mg2+, Cu2+, Ni2+, Zn2+ and Pb2+ ion were investigated. H2BR can form expanded and stable monolayer on neutral and acidic subphases, while it can hardly form monolayer on basic subphases. The acid-base equilibrium of H2BR was discussed at the air-water interface. The association-dissociation of H2BR with H+ ions in the interfacial region was very different from that in the bulk solution. Some information regarding the packing density and degree of ionization of the head group under different experimental conditions were obtained. The formation of H2BR-metal complexes leads to changes in the shape of isotherms of H2BR and changes in UV-visible absorption spectra of the monolayer assemblies. In XPS spectra new XPS peaks assigned to the metal ions containing subphases appeared. Low-angle X-ray diffraction indicates that a Y-type LB films were formed with bilayer spacing of ca. 2.50 nm.