Agarose is a natural seaweed polysaccharide and widely used in the medicine, food, and biological fields because of its high gel strength, non-toxicity, and electrical neutrality. The sulfate group is one of the main charged groups that affect the performance of agarose. In the present study, a simple, eco-friendly, and efficient method was explored for agarose preparation. After desulfation with hydrogen peroxide (H2O2), the sulfate content of agar reached 0.21%. Together with gel strength, electroendosmosis, gelling and melting temperature, the indicators of desulfated agar met the standards of commercially available agarose. Notably, the desulfated agar can be used as an agarose gel electrophoresis medium to separate DNA molecules, and the separation effect is as good as that of commercially available agarose. Further, the H2O2 desulfation process was analyzed. The addition of a hydroxyl radical (HO•) scavenger remarkably decreased the H2O2 desulfation rate, indicating that HO• has a certain role in agar desulfation. Sulfate content detection indicated that sulfur was removed from agar molecules in the form of sulfate ions (SO42-) and metal sulfate. The band absence at 850 cm-1 indicated that the sulfate groups at C-4 of D-galactose in sulfated galactan were eliminated.