AbstractP. aeruginosa, a leading nosocomial pathogen, commonly causes chronic biofilm infections in tissues and biomedical devices, including wound infections, osteomyelitis, and infective endocarditis, heavily threatening life. The dynamic lifecycle of these biofilms leads to persistent generation, making it challenging to prevent and disperse these biofilms effectively. Herein, a topological eight‐arm zwitterion‐conjugated glycomimetics (PCBAA‐b‐PLAMA)8 to address this challenge by exerting a dual‐blocking effect on P. aeruginosa biofilms is introduced. Initially, carboxybetaine acrylate (CBAA) and 2‐lactobionamidoethyl methacrylate (LAMA) are introduced to the topological bromine‐based initiator to prepare (PCBAA‐b‐PLAMA)8. This copolymer demonstrats remarkable efficiency in dispersing P. aeruginosa biofilms, approximately up to 99%. This high efficacy can be attributed to the multivalent and triaxial interactions between LAMA and CBAA groups, which enable the capture of P. aeruginosa cells and the biofilm matrix. Furthermore, (PCBAA‐b‐PLAMA)8 efficiently inhibit the expression of resistance genes related to biofilm formation and antibiotic efflux, including cdrA, lasB, mexE, and mexH, regaining bacterial cell sensitivity to antibiotics and further facilitating the natural dispersal of biofilms. This study provides a generic dual‐blocking strategy for the efficient eliminating of biofilms from biomedical devices.