In this study, a novel polystyrene-block-quaternized polyisoprene amphipathic block copolymer (PS-b-PIN) is derived from anionic polymerization. Quaternized polymers are prepared through post-quaternization on a functionalized polymer side chain. Moreover, the antibacterial activity of quaternized polymers without red blood cell (RBCs) hemolysis can be controlled by block composition, side chain length, and polymer morphology. The solvent environment is highly related to the polymer morphology, forming micelles or other structures. The polymersome formation would decrease the hemolysis and increase the electron density or quaternized groups density as previous research and our experiment revealed. Herein, the PS-b-PIN with N,N-dimethyldodecylamine as side chain would form a polymersome structure in the aqueous solution to display the best inhibiting bacterial growth efficiency without hemolytic effect. Therefore, the different single-chain quaternized groups play an important role in the antibacterial action, and act as a controllable factor.