<p>Native fucosylated glycosaminoglycan (FG), a structurally unique polysaccharide with chondroitin sulfate like backbone and sulfated fucose side chains, has multiple anticoagulant mechanisms. Analyzing the interaction between FG and coagulation proteins will help to further understand its pharmacological mechanisms. Previously, we have reported that high molecular weight depolymerized FG (dFG) and unfractionated heparin (UFH) exhibit different binding models for antithrombin (AT), which is a primary natural anticoagulant in plasma by inhibiting coagulation proteases including thrombin (FIIa). In this study, the effect of dFG or UFH on the interaction between AT and FIIa was detected by biolayer interferometry. The relative AT binding affinity and AT-dependent anti-FIIa activity of dFG derivatives were measured to elucidate the structure-activity relationship. The results demonstrate that high molecular weight dFG significantly promotes the irreversible binding of FIIa and AT by a template mechanism. As the molecular weight decreases, the relative AT binding affinity of dFG decreases and molecular weight above 8.55&#xa0;kDa is required for their interaction in this study. Both the fucose side chains and carboxyl groups are indispensable for dFG to bind with AT and inhibit FIIa activity. This study clarifies the mechanism and structure-activity relationship of dFG inhibiting FIIa by AT, providing references for the development of novel anticoagulant drugs based on FG and its derivatives.</p>