In order to get concentrated distribution of small molecular hyaluronan, 6g/L of high molecular hyaluronic acid aqueous solution was prepared, added with a certain gradient of hyaluronidase, and controlling the hydrolysis time. The results showed that controlling the amount of hyaluronidase and hydrolysis time, particular molecular weight hyaluronic acid could be prepared according to the law of the hyaluronan molecular weight decreased,which included tetrasaccharide and hexasaccharide. Furthermore,through high performance size exclusion chromatography and agarose gel electrophoresis, we could prove that the molecular weight distribution of hydrolysate was concentrated.