Heparan sulfates (HSs) are the main components in the glycocalyx which covers endothelial cells and modulates vascular homeostasis through interactions with multiple Heparan sulfate binding proteins (HSBPs). During sepsis, heparanase increases and induces HS shedding. The process causes glycocalyx degradation, exacerbating inflammation and coagulation in sepsis. The circulating heparan sulfate fragments may serve as a host defense system by neutralizing dysregulated Heparan sulfate binding proteins or pro-inflammatory molecules in certain circumstances. Understanding heparan sulfates and heparan sulfate binding proteins in health and sepsis is critical to decipher the dysregulated host response in sepsis and advance drug development. In this review, we will overview the current understanding of HS in glycocalyx under septic condition and the dysfunctional heparan sulfate binding proteins as potential drug targets, particularly, high mobility group box 1 (HMGB1) and histones. Moreover, several drug candidates based on heparan sulfates or related to heparan sulfates, such as heparanase inhibitors or heparin-binding protein (HBP), will be discussed regarding their recent advances. By applying chemical or chemoenzymatic approaches, the structure-function relationship between heparan sulfates and heparan sulfate binding proteins is recently revealed with structurally defined heparan sulfates. Such homogenous heparan sulfates may further facilitate the investigation of the role of heparan sulfates in sepsis and the development of carbohydrate-based therapy.