Abstract: Ethanol is produced in high concentration of 75 mg/ml or above such as 200 mg/ml by using an immbolized ethanol-producing microorganism to convert sugar to ethanol in a nutrient culture broth containing not more than 100 mg/ml sugar until the concentration of sugar is not more than 20% of its initial concentration, then adding a fresh culture broth containing not less than 100 mg/ml sugar and converting sugar to ethanol with the immobilized microorganism to produce the desired high concentration of ethanol. The microorganism is preferably immobilized in a sulfated polysaccharide gel as a dense layer near the surface of the gel.