Multilayer film elements used in hospitals for clinical diagnosis contain all chemicals that are necessary for colorimetric reaction sequence including enzymes. Because of the nature of protein, most enzymes show less stabilities than other organic or inorganic compounds. Therefore, it is very important to prevent the loss of enzyme activity in multiplayer film elements. So for, some sugars are known to stabilize enzymes and recently one of disaccharides called trehalose has been the topic in many fields such as cosmetics and food industries. Here we demonstrate that sugars like trehalose, maltose and sucrose stabilize enzymes also in multiplayer film elements. Multilayer film elements consist of transparent support, reaction layer that is made of gelatin, and blood spreading layer. Enzymes are usually immobilized in reaction layer or spreading layer. In this paper, we report the stabilization of enzymes immobilized on the surface of fiber as spreading layer by using some sugars. Those three disaccharides clearly protect enzymes from the loss of their activities during the manufacturing process and the storage of multiplayer film elements. And we also refer about the possibility of Km value as the index of the stability of enzymes in aqueous solution including many kinds of chemicals.