The accurate protein-protein separation is important but technically challenging. Achieving such a precise separation using membrane requires the selective channels with appropriate pore geometry structure and high anti-fouling property. In this study, polyethersulfone-b-poly(sulfobetaine methyl methacrylate) (PES-b-PSBMA) was synthesized and engineered onto polysulfone (PSF) ultrafiltration (UF) membrane to fabricate zwitterionic nanospheres engineered co-polymer (ZN-e-CoP) composite membrane via dynamic self-assembly micelle deposition. On the one hand, self-assembly zwitterionic nanospheres were used as blocks to construct hydrophilic layers with size-dependent sieving channels, endowing ZN-e-CoP composite membranes with enhanced permselectivity and protein-protein separation abilities, meanwhile zwitterionic groups from nanospheres reinforced the structure stability of nanospheres/nanospheres and nanospheres/membrane via multiple intermolecular interactions. On the other hand, zwitterionic nanospheres can induce to produce the hydration layer enveloping themselves by binding water molecules, where hydration layer acts as a protective barrier on the membrane surface, impeding the protein adhesion. Hence, ZN-e-CoP_1a composite membrane exhibited superior separation properties with Lysozyme/Bovine Serum Albumin (BSA) separation factor of 18.1 and 95.4 % rejection against BSA, 10.1 and 2.3 times, respectively, higher these of pristine PSF membrane (1.8 and 42.1 %), without obviously sacrificing water flux. Simultaneously, hydration layer enables the ZN-e-CoP_1a membrane with enhanced anti-fouling performance and durability during the long-term operations. The proposed approach opens new pathways to fabricate excellent anti-fouling membranes for precise protein-protein separation.