Abstract: The structure of heparan sulfate determines its interaction with proteins, therefore the analysis of heparan sulfate can help further clarify structure and function relationships. The characteristic fragmentation pattern of twelve heparan sulfate disaccharide was discussed by electrospray ionization-ion trap mass spectrometry （ESI-MSⁿ） in negative mode. The results show that the position and quantity of sulfate groups in disaccharide determine its stability in MS, moreover, C—2 sulfate and C—6 sulfate in disaccharide are more stable than N-sulfate. MS² revealed that the disaccharide isomers have a characteristic fragment pattern. The results may contribute to identify the disaccharides isomer of heparan sulfate, which can provide theoretical knowleage to identify the structural of heparan sulfate.