A one-pot strategy was applied to synchronize enzymatic monomer transformation with reversible addition fragmentation chain transfer (RAFT) polymerization for the synthesis of glycopolymers with highly branched gradient architectures. Also, the linear analogues, block glycopolymers, and gradient glycopolymers were also synthesized for comparison. The binding ability of glycopolymers toward bacteria was then studied by optical density (OD) test, confocal laser scanning microscopy (CLSM), and quartz crystal microbalance with dissipation (QCM-D). The results show that the highly branched gradient glycopolymers have the most remarkable bacteria-binding ability compared with the two linear analogues, gradient glycopolymers, and block glycopolymers. The highly branched glycopolymers were further used as inhibitors in the anti-infection test, demonstrating a significant inhibitory effect on preventing bacteria from infecting the cells.