Hyperbranched polymer brushes with a three-dimensional dendritic structure are used in antifouling applications to obtain bioinert and compact dendritic structures. Though hyperbranched polyglycerol (HPG) is extensively utilized in the antifouling layer, there is still a lack of direct studies on the relationship between the interfacial properties and topology effect of hyperbranched polymer brushes. Here, we established the degree of chain volume overlap ( D v ) to characterize the spatial shielding efficiency generated by HPG brushes and investigated the impact mechanism of the variable chain length on the interfacial physicochemical properties. The results revealed the D v -relevant feature of performance that the most densely packed HPG brushes for a medium-length L HPG3.07 enable the functional surface to display optimal antifouling performance toward protein adsorption by forming the most effective space barrier and hydrated layer in appropriate molecular weights and graft density. Moreover, we clarified the advance of hyperbranched polymer brushes exhibited in topology effects for imparting surface-enhanced resistance to biofouling relies on the generable higher steric hindrance as compared with linear analogs. This study established a D v -relevant evaluation model for acquiring an optimized antifouling surface based on the appropriate choice of polymer structure, topology morphologies, and grafting parameters.