As one of the seaweed polysaccharide, agarose has received much attention because of its biocompatibility. However, its application in biomedical field was limited with its biological inertia. Modification with some functional groups is needed to obtain agarose derivatives with biological activity and expand its applications. Consequently, agarose was sulfated with chlorosulfonic acid-pyridine with formamide as dispersing agent. The orthogonal test result showed that the optimal reaction condition was the reaction time being 4 h, the reaction temperature 65 ℃, and the ratio of chlorosulfonic acid to agarose 1-4(mL/g). Two kinds of the insoluble agarose sulfate (below 37 ℃) were synthesized with degree of substitution (DS) being 0.17 and 0.43 respectively. Infrared spectroscopy (IR) and 13C nuclear magnetic resonance (13C-NMR) spectroscopy results proved that C3-6 in agarose was sulfated. Their hydrophobic property and BSA adsorption capacity rose with increasing DS, while the adsorption of Hb was reduced. The anti-clotting properties of agarose sulfate were significantly improved, and agarose sulfate could protect red blood cells from deformation after adsorption of BSA. These findings demonstrate that the cold-water insoluble agarose sulfate has a promise for applications as heparin-like material in anticoagulation or endothelial regeneration scaffold. As one of the seaweed polysaccharide, agarose has has received much attention because of its biocompatibility. However, its application in biomedical field was limited with its biological inertia. Modification with some functional groups is needed to obtain agarose derivatives with biological activity and expand its applications. The orthogonal test result showed that the optimal reaction condition was the reaction time being 4 h, the reaction temperature 65 ° C, and the ratio of chlorosulfonic acid to agarose 1- Infrared spectroscopy (IR) and 13C nuclear magnetic resonance (13C-NMR) spectroscopy were performed at 4 (mL / g). Two kinds of the insoluble agarose sulfate (below 37 ° C) were synthesized with a degree of substitution results proved that C3-6 in agarose was sulfated. Their hydrophobic property and BSA adsorption capacity rose with increasing DS, while the adsor The anti-clotting properties of agarose sulfate were significantly improved, and agarose sulfate could protect red blood cells from deformation after adsorption of BSA. These findings demonstrate that the cold-water insoluble agarose sulfate has a promise for applications as heparin-like material in anticoagulation or endothelial regeneration scaffold.