Abstract: Sea cucumber saponin Echinoside A was hydrolyzed into low hemolytic secondary glycosides by commercial enzymes. In order to obtain less hemolysis from the rare secondary sea cucumber saponins, sea cucumber glycoside-Echinoside A was hydrolyzed by 5 glycosidases. The glycosidases with enzymatic hydrolysis effects for EA were screened out by TLC, HPLC-MS and ESI-MS/MS, and the enzymatic products structures of which were identified and hemolytic toxicity studies were performed. The results showed that EA could be hydrolyzed by Pectinase, Pectinex Ultra SP-L and Celluclast 1.5L enzymes. The enzymatic hydrolysates of the Pectinase were secondary sea cucumber saponin-Compound 1 with two saccharide groups and secondary sea cucumber saponin-compound 2 with one saccharide group;The enzymatic hydrolysates of the Pectinex Ultra SP-L and Celluclast 1.5 L were secondary sea cucumber saponin-Compound 1.The other two enzymes had no enzymatic hydrolysis effect. Hemolysis test showed that the hemolytic toxicity of the products was obviously reduced. The hemolysis of compound 2 was remarkably reduced to less than 30%.