The dTDP-D-glucose 4,6-dehydratase from Salmonella enterica was immobilized using covalent binding to cyanogen bromide activated sepharose. The immobilized enzyme was used to produce dTDP-4-keto-6-deoxy-D-glucose, a key sugar intermediate that can be used economically to produce diverse classes of unusual sugars appended in various antibiotics. The enzyme was immobilized on the sepharose after activation with cyanogen bromide. The maximum immobilization (80.03％) was achieved after 14 h of coupling. The covalently immobilized enzyme was stable, and an average of 78.4 ％ conversion was achieved until 120 h of immobilization when it was repeatedly used. Similar conversion was noticed for the first batch using the enzyme entrapped-hydrogel but activity was gradually decreased in the following batches. The production of dTDP-4-keto-6-deoxy-D-glucose by using an immobilized enzyme has high potential for commercial application.