Ultrathin films of α-helical polyglutamate with long flexible side chains (PG) were fabricated by the LB technique, and their nanostructure was investigated by the energy transfer method. The rodlike main chain of PG was labeled by incorporating either energy donor (naphthalene, Np) or acceptor (anthracene, An) molecules via short side chains. The fluorescence spectra of Np−An multilayers with the same number of spacing layers gave different energy transfer efficiencies, depending on the mode of depositions. This phenomenon was interpreted as being caused by the noncentered structure of the polymer backbone, which was probed by both X-ray reflectometry and fluorescence spectroscopy. The Y-type deposition induced a bilayer structure in which the stiff main chains of each monolayer were located close to the center of the bilayer. The layered structures showed excellent thermal stability up to around 120 °C. By thermal treatment higher than 120 °C, the layered structure relaxed to a stable state in which the α-helical backbones in each layer became intermingled.