Provided are a sulfated heparin oligosaccharide as well as a preparation method and an application thereof. The sulfated heparin oligosaccharide is characterized in that a non-reducing end of a molecule of the sulfated heparin oligosaccharide contains an unsaturated double bond resulting from enzymolysis by heparinase, as well as a uronic acid derivative and a glycosylamine derivative; the sulfated heparin oligosaccharide has a structure represented by formula I, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R a , R b , R c and R d are independently SO 3 - or H; R x ', R y ' and R z ' are independently COCH 3 or SO 3 - , and n is 1-3. The preparation method obtains a sulfated oligosaccharide with a controllable degree of sulfation. The sulfated heparin oligosaccharide has a high activity for inhibiting heparanase in vitro, with an activity 4-5 times higher than that of heparin for inhibiting cell adhesion and migration, and an activity 2-3 times higher than that of heparin for resisting
