This paper reports the supramolecular chirality from optically inactive polymers through an interfacial organization at the air/water interface. A series of poly(p-phenylenevinylene) (PPV) derivatives with dimethyloctyloxyphenyl substitutents were designed, and their spreading Langmuir monolayers as well as Langmuir-Schaefer (LS) films were investigated. The films were characterized by the p-A isotherm, UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy. The p-A isotherms showed plateau regions for all the polymers. The monolayers of the polymers can be transferred onto solid substrates as multilayer films, in which chromophores exhibited an H-aggregation. Although no CD signal was detected in both chloroform solutions and the cast films, supramolecular chirality was observed for some of the polymers at certain conditions. The larger steric hindrance of the side chain in the PPV polymer and a lateral compression of the Langmuir film over the plateau region in the p-A isotherm were necessary to obtain the chiral multilayers. It was suggested that the supramolecular chirality of the films was due to the formation of a stereoregular arrangement of the side chain along the polymer backbone through an interfacial organization.