Polymer brushes with different topological architectures exhibit unparalleled interfacial and physicochemical properties and are being widely utilized in antifouling applications. However, there is an absence of a thorough understanding of the antifouling process of dynamic flow mediated by the topological structure of polymer brushes. Here, it is highlighted how the interface parameters related to biofouling in flowing carrier fluid are tuned by topologically different architectures. The mechanism by which three brushes with various topological structures (cyclic, looped, and linear brushes) encounter biological media was revealed by relating protein adhesion with nanomechanics and protein conformational transitions on poly(2-ethyl-2-oxazoline) (PEtOx) brushes. In contrast to the classically linear analogue, the cyclic PEtOx brushes confered an enhanced steric barrier and excellent lubrication at the critical density region. The impenetrable and smoother layer prevented the approach and shortened the residence time for protein on the surface, providing optimal antifouling properties at low shear rates. The looped brushes also significantly inhibited protein adhesion under prolonged high shear rates due to their unshakable conformational characteristics. These findings detailed a new evaluation framework behind polymer brushes of topology-driven biofouling repulsion under flow conditions and pointed the way toward a promising approach for the effectiveness of biomaterial design.