An immobilized enzymatic active substance having an improved stability of enzymatic activity is prepared which comprises an enzymatic active substance such as microbial cells entrapped within a gel matrix of an unsubstituted amino, monoalkylamino or dialkylamino group-introduced polysaccharide having not less than 10 w/w % of sulfate moiety in the molecule thereof such as carrageenan. The immobilized enzymatic active substance is prepared by admixing the enzymatic active substance and an aqueous solution of the polysaccharide and then allowing to form a gel of the polysaccharide.