A　method　for　the　preparation,　purification　and　sequence　analysis　of　heparin　oligosaccharides　with　internal　N-acetylated　glucosamine　residues　was　established.　Firstly,　low　molecular　weight　heparin　was　completely　digested　by　heparinase　I,　and　separated　by　Bio-Gel　P-10　gel　filtration　chromatography.　A　range　of　oligosaccharides　from　dp2　up　to　dp14　was　obtained.　Four　of　dp6　and　three　of　dp8　oligosaccharides　with　N-acetylated　residues　were　purified　by　strong　anion　exchange　(SAX)　high　performance　liquid　chromatography　(HPLC).　Secondly,　the　disaccharide　compositions　of　the　purified　oligosaccharides　were　analyzed　by　SAX-HPLC　after　combined　digestion　using　heparinase　I,　II　and　III.　The　sequence　of　oligosaccharides　was　deduced　according　to　its　disaccharide　composition　and　the　substrate　specificity　of　heparinase　I.　The　sequencing　results　showed　that　the　N-sulfated　disaccharides　were　internal,　with　the　terminal　disaccharides　being　predominantly　highly　sulfated,　as　expected　from　the　substrate　specificity　of　heparinase　I.　Finally,　the　purified　oligosaccharides　were　characterized　by　electrospray　ion　trap　time.　of.　flight　mass　spectrometry　(ESI-IT-TOF-MS)　system　in　the　negative　ion　model.　The　results　showed　that　many　of　the　-SO32-　groups　were　lost　in　ion.　source.　The　major　ion　fragments　of　dp6　oligosaccharides　had　2　and　3　negative　charges,　while　dp8　oligosaccharides　had　2　to　5　negative　charges.　The　main　fragmentation　of　dp6　appeared　in　glycosidic　linkage,　the　X-0,X-2　fragmentation　existed　in　N-acetylgalactosamine　while　the　(0,2)Z　one　appeared　in　glucuronic　acid.　This　strategy　offers　possibilities　for　the　sequencing　of　heparin　oligosaccharides　with　rare　structure.