The invention relates to O-unsaturated fatty acid acylated chitosan oligosaccharides, which are characterized in that the O-unsaturated fatty acid acylated chitosan oligosaccharides are a mixed component and have a molecular weight of 800-25000 Da; the deacelation degree of the mixed component is 50-100%; the substitution degree of O-unsaturated fatty acid is 30-200%; and the components of the O-unsaturated fatty acid acylated chitosan oligosaccharides have a structure shown in the specification, wherein R1 is H or CH3CO-, R2 is C12-22 unsaturated fatty acid acyl or H, and n is 0-30. The O-unsaturated fatty acid acylated chitosan oligosaccharides have activities of resisting oxidation, delaying aging, inhibiting tumor, resisting inflammation, lowering blood pressure, preventing thrombosis, improving diabetes mellitus, lowering blood sugar, resisting atherosclerosis and the like, and can be used as important raw materials for medicines, health-care food and cosmetics.