In this study, polypeptide/polysaccharide graft copolymers pullulan-g-poly(L-lysine) (PULPLL) was prepared through the ring opening polymerization of ε -benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of pullulan. The PULPLL copolymer nanoparticles, which are prepared by the diafiltration method, were investigated by using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectra (1H NMR), dynamic light scattering (DLS), transmission electron microscope (TEM), and fluorescence spectroscopy. The PULPLL copolymers formed self-aggregated nanoparticles in the aqueous milieu. The physicochemical characterization revealed that the nanoparticle size ranged between 60 and 500 nanometers, and the critical aggregation concentration (CAC) is 0.001~0.1 g/L, depending on the degree of substitution. With negligible cytotoxicity and higher transfection efficiency, PULPLL nanoparticles may be safe gene carriers.