Abstract: Triblock copolypeptides poly(L-lysine-N_ε-2-2-(2-methoxyethoxy)ethoxyacetyl)-b-poly(L-glutamate-γ-benzyl)-b-poly(L-lysine-N_ε-2-2-(2-methoxyethoxy)ethoxyacetyl) (PLys-(EG)₂-b-PBLG-b-PLys-(EG)₂) and poly(L-glutamate-γ-benzyl)-b-poly(L-lysine-N_ε-2-2-(2-methoxyethoxy)ethoxyacetyl)-b-poly(L-glutamate-γ-benzyl) (PBLG-b-PLys-(EG)₂-b-PBLG) were synthesized via N-carboxyanhydride (NCA) ring opening polymerization using N, N'-(disulfanediylbis(ethane-2, 1-diyl))bis(1, 1, 1-trimethylsilanamine) (N-TMS) as the initiator. The hydrophilic block poly(L-lysine-N_ε-2-2-(2-methoxyethoxy)ethoxyacetyl) (PLys-(EG)₂) and hydrophobic block poly(L-glutamate-γ-benzyl) (PBLG) maintained stable α-helix conformation in water and N, N-dimethylformamide (DMF) mixture. The a-helices were in rigid rod-like constellation and inflexible in self-assembly. Therefore, the amphiphilic copolypeptides were expected to show distinct self-assembly behavior in selective solutions. Herein, DMF-water mixture was adopted as the media and copolypeptides were allowed self-assembly through traditional strategies. ¹H-Nuclear Magnetic Resonance (¹H-NMR) and Gel Permeation Chromatography (GPC) confirmed the formation of desired copolypeptides. The assemblies were observed with Transmission Electron Microscope (TEM). Results demonstrated that the obtained polypeptides showed controllable molecular weights (M_w) and narrow polydispersity index (PDI). PLys-(EG)₂-b-PBLG-b-PLys-(EG)₂ self-assembled into small micelles while containing long hydrophilic PLys-(EG)₂ blocks, otherwise only spherical and rod-like large compound micelles with different dimensions were observed. In contrast, PBLG-b-PLys-(EG)₂-b-PBLG self-assembled into larger micelles while containing long hydrophilic blocks, which showed unstableness while the hydrophobic blocks turned longer. The unusual all-rigid skeletons and the confined conformation of the amphiphilic triblock copolypeptides might be the reason of these results.