Amphiphilic random copolymers of poly(gamma-glutamic acid)-graft-L-phenylalanine (gamma-PGA-Phe), with various lengths of gamma-PGA main chains (molecular weight of 70, 140, and 220 kDa) plus hydrophobic moieties of Phe groups (grafting degree 12-60%), self-assembled in aqueous media to form nanoparticles (NPs). The aggregation number (N-agg) could be adjusted according to their molecular structures as well as the preparative methods/conditions. The gamma-PGA-Phe NPs were further characterized by means of dynamic and static light scattering, small-angle neutron scattering, as well as steady-state fluorescence measurements/quenching techniques. The single chain state had hydrophobic domains, and formed spherical structures, herein called unimer NPs, and were obtained using molecular weights of gamma-PGA higher than 140 kDa conjugated with Phe at 27-42% due to the balance of hydrophobicity/hydrophilicity along the single polymer chain. The number of hydrophobic domains in one NP (N-domain), estimated by means of fluorescence quenching techniques and the rigidity of the inner particles detected by dipyrene fluorescence demonstrated that the N-domain and the rigidity were affected by the particle size and preparative methods. In addition, the effect of pH on the stability of the unimer NPs indicated a reduction of the N-domain upon the pH, supporting a loose packing due to hydrophobic association under alkaline conditions.