Abstract: 6-O-carboxymethyl chitin (6-O-CMCH) was prepared from chitin at low temperature, and then 6-O-CMCH was deacetylated to obtain 6-O-carboxymethyl chitosan (6-O-CMCS). The sulfated 6-O-CMCS with limited controllable substitution was prepared by a controlled reaction between 6-O-CMCS and chlorosulfonic acid/concentrated sulfuric acid. 6-O-CMCH and sulfated 6-O-CMCS were characterized with conductometric titration, elemental analysis, FT-IR, and 1H-NMR methods. The sulfated 6-O-CMCS was measured to determine its activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). Results indicated that the structure and blood anticoagulation of sulfated 6-O-CMCS were similar to that of heparin, and its activity of blood anticoagulation was mainly due to the suppression of endogenous and common coagulation pathway.