A method for the synthesis of novel disaccharides was developed. It involved the following two steps. The first step consisted of two continuous reactions: the conversion of maltose to beta-D-glucose-1-phosphate and D-glucose by the phosphorolytic activity of maltose phosphorylase and the specific consumption of only D-glucose by incubation with glucose-consuming yeast cells. The second step involved the addition of an appropriate carbohydrate and its condensation with the remaining beta-D-glucose-1-phosphate by the synthetic activity of maltose phosphorylase or trehalose phosphorylase. Several factors affecting the yields of disaccharides were optimized. Using this method, five maltose-like derivatives and two trehalose-like derivatives were synthesized from maltose and the corresponding carbohydrates. Among these, 4-O-alpha-D-glucopyranosyl-L-fucopyranose (Glc(alpha1-4)Fuc) and alpha-d-glucopyranosyl alpha-D-fucopyranoside (Glc(alpha1-1alpha)Fuc) were purified, and identified by 1H NMR and 13C NMR.