Glucosinolates can be hydrolyzed by the enzyme commonly known as myrosinase (E.C. 126.96.36.199)to a variety of biological compounds. Myrosinase (MYR)has been immobilized through the flexible spacers of different length on cross-linked chitosan resin (CCR). Ethylene diamine, hexamethylenediamine and decamethylene-diamine have been separately used as spacers. The influence of the flexible spacer length on the immobilized MYR (IMYR)properties were evaluated. The optimum pH and V max of IMYR linearly increase with the flexible spacer length, and the optimum temperature and K m of IMYR show an opposite trend. The recyclability of IMYR was good, with 90% recovery of activity after 10 cycles and 80% recovery after 30 cycles. IMYR was highly stable under storage conditions, with 95% recovery of activity after one year storage at 4 °C. The IMYR with the longest flexible spacer, decamethylene-diamine, was used as a biocatalyst for sulforaphene production. The overall hydrolysis ratio of glucoraphenin was 93.25 ± 0.91% and the activity of DDMCCR-IMYR remained 95% after 10 days of continuous use.