We have prepared for the first time an organized monolayer with long-chain amphiphiles possessing an isothiouronium segment. Interactions between the monolayer and anions in aqueous subphase have been investigated and show a significant selectivity of H2PO4-. Also, by fabricating the Langmuir-Blodgett (LB) films under competitive conditions of the anions used and picrate as an indicator, we can read out H2PO4- recognition using UV-vis spectroscopy, providing a potential application for the design of film-based anion sensor systems.