The invention provides sulfated heparin oligosaccharide as well as a preparation method and application thereof. The sulfated heparin oligosaccharide is characterized in that a non-reducing end of a sulfated heparin oligosaccharide molecule contains an unsaturated double bond produced through enzymolysis of heparinase as well as uronic acid derivatives and glycosylamine derivatives; the sulfated heparin oligosaccharide has a structure represented by a formula I, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, Ra, Rb, Rc and Rd are independently SO3<-> or H; Rx', Ry' and Rz' are independently COCH3 or SO3<->, and n is 1-3. The sulfated heparin oligosaccharide with controllable sulfating degree prepared by virtue of the preparation method has very high activity for inhibiting heparanase in vitro, the activity of the sulfated heparin oligosaccharide for inhibiting cell adhesion and migration is 4-5 times higher than that of heparin, and the activity for inhibiting tumor metastasis in mice is 2-3 times higher than that of the heparin; the sulfated heparin oligosaccharide has relatively good tumor metastasis resistance and relatively high specificity.