Surface-grafted peptide nanospheres consisting of hydrophobic poly(L-phenylalanine) with hydrophilic poly(ethylene glycol) (PEG) grafts were successfully prepared by the one-step polymerization of L-phenylalanine N-carboxyanhydride with the dual initiators of hydrophobic n-butylamine and hydrophilic NH2-monoterminated PEG (NH2-PEG). The monodispersed peptide nanospheres were stably self-assembled during polymerization in a mixture of water/dimethyl sulfoxide to create a colloidal solution, but only aggregated in water or organic solvents. When n-butylamine or NH2-PEG was used as a solitary initiator, the peptide nanospheres were not formed. The peptide nanospheres showed high dispersion-stability in water, and their diameter was approximately 300 nm. Furthermore, the peptide nanospheres were well-redispersed in water, retaining the same diameter and monodispersity even after lyophilization. Peptide nanospheres with the functional carboxylic acid on their graft layer were also successfully prepared by the one-step preparation method. This one-step preparation method of surface-grafted peptide nanospheres will be useful as an advanced technology to develop biodegradable functional nanospheres.