Heparinase from different sources can eliminate heparin or/and heparan sulfate into various low-molecular weight heparins with different characteristics. Porcine intestinal mucosa heparin was degraded into a series of oligosaccharides by a novel heparinase from the species Sphingobacterium. Disaccharide components from the digests were separated and purified by ultrafiltration and HPLC. Five major peaks appeared as three types according to their retention time. The mass spectrometry of peak I mainly gave the non-sulfated disaccharide with the mass of 379 Da. Peak II and III were indicated as two major monosulfated disaccharides with molecular mass of 417 and 459 Da respectively. Moreover, the peak III represented an N-acetyl disaccharide. Both peak IV and V showed the same mass of 496 Da, hinting that they were disulfate-substituted disaccharides. No trisulfate-substituted disaccharides were detected in the mixture of the heparin digest though they were abundant in the heparin structure. The results revealed that the heparinase might specifically cut the sites with low sulfated domain in heparin.