As the hydrolysis product of radish thioglucoside catalyzed by thioglucosidase, sulforaphene (SFE) is a kind of functional ingredient with broad market prospects in virtue of its strong anti-oxidation, anti cell mutation, anti-cancer, antibacterial and herbicidal activities. Due to the fact that free thioglucosidase can be only used once, researching immobilized thioglucosidase and its enzymatic properties has determinant impact on the practical preparation of SFE. However, current immobilized thioglucosidase unexceptionally suffer from serious limitations of low mechanical strength and decomposition by microorganisms, not enough to support the enzyme to achieve continuous industrial reaction. In this study, thioglucosidase was separated and purified from radish seeds by means of extraction, precipitation and membrane separation, so the specific activity of thioglucosidase was increased to 8 times of the original. Then the purified thioglucosidase was covalently immobilized on amine-based resin by glutaraldehyde and tests have been performed on the determination of optimal cross-linking time, optimal cross-linking pH and the molar ratio of free enzyme, glutaraldehyde and carrier amino groups. Therewith the immobilized thioglucosidase was proved to possess superior enzymatic properties including pH adaptation, temperature adaptation, thermal stability, reusability and storage stability, compared with the free thioglucosidase. After the optimal substrate concentration and flow rate was found, the yield of SFE produced by the immobilized thioglucosidase reactor column reached 9.10%, and the benefit of immobilized thioglucosidase is 2.91 times that of direct fermentation. The immobilized thioglucosidase on amine-based resin can be further implemented for the production of SFE through enzymatic method instead of chemical synthesis or direct fermentation. (C) 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.