A novel sorbitol dehydrogenase having excellent substrate affinity and substrate specificity which can be used to measure D-sorbitol which occurs in human erythrocytes and serum in a slight amount or D-sorbitol or D-fructose contained in foods, a microorganism for producing such an enzyme, and a process for the production of the enzyme using such a microorganism. Also disclosed is provides a method for the measurement of sorbitol using the foregoing sorbitol dehydrogenase and a reagent for the quantitative determination therefor.