The preparation of alkyl chain-grafted poly(l-lysine) (PLL) vesicles with tunable molecular assembly in aqueous solution and the evaluation of their membrane permeability by drug release experiments have been investigated. Upon grafting long alkyl chains, polypeptides confined in the assembled nanostructures adopted ordered conformations such as α-helices or β-sheets/turns, leading to the dense packing of membranes and, consequently, the decreases in vesicular size and membrane permeability. The vesicles can also be cross-linked by genipin to form stable structures with tunable membrane permeability. Additionally, these vesicles exhibited noticeable pH-sensitive behavior, depending on the grafted alkyl chain and cross-linking.