Abstract: A high performance liquid chromatographic ( HPLC) method for simultaneous determination of uronic acids, amino sugars and neutral sugars in polysaccharide from sea cucumber by pre- column derivatization with1-phenyl-3-methyl-5-pyrazolone ( PMP) was developed. The polysaccharide was hydrolyzed by the 2mol /L trifluoroacetic acid, and then derivatived with PMP. The uronic acids, amino sugars and neutral sugars were separated well by reverse- phase HPLC. The results showed that glycosaminoglycan consisted of mannose, galactose, fucose, glucosamine, galactosamine, glucuronic acid.The HPLC method is simple, rapid, sensitive, and convenient.