Amphiphilic block or graft copolymers have been demonstrated to form a variety of self-assembled nano/microstructures in selective solvents. In this study, the self-association behavior of biodegradable graft copolymers composed of poly(gamma-glutamic acid) (gamma-PGA) as the hydrophilic segment and L-phenylalanine (Phe) as the hydrophobic segment in aqueous solution was investigated. The association behavior and unimer nanoparticle formation of these gamma-PGA-graft-Phe (gamma-PGA-Phe) copolymers in aqueous solution were characterized with a focus on the effect of the Phe grafting degree on the intra-and interpolymer association of gamma-PGA-Phe. The particle size and number of polymer aggregates (N-agg) in one particle of the gamma-PGA-Phe depended on the Phe grafting degree. The size of gamma-PGA-Phe with 12, 27, 35, or 42% Phe grafting (gamma-PGA-Phe-12, -27, -35, or -42) was about 8-14 nm and the N-agg was about 1, supporting the presence of a unimolecular graft copolymer in PBS. The pyrene fluorescence data indicated that gamma-PGA-Phe-35 and -42 have hydrophobic domains formed by the intrapolymer association of Phe attached to gamma-PGA. These results suggest that the Phe grafting degree is critical to the association behavior of gamma-PGA-Phe and that gamma-PGA-Phe-35 and -42 could form unimer nanoparticles. Moreover, when gamma-PGA-Phe-42 dissolved in DMSO was added to various concentrations of NaCl solution, the particle size and N-agg could be easily controlled by changing the NaCl concentration during the formation of the particles. These results suggest that biodegradable gamma-PGA-Phe is useful for the fabrication of very small nanoparticles. It is expected that gamma-PGA-Phe nanoparticles, including unimer particles, will have great potential as multifunctional carriers for pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.