S-(+)-2,2-dimethylcyclopropanecarboxylic acid (S-(+)-DMCPA) is a key chiral intermediate for the synthesis of Cilastatin. The enzymatic preparation of S-(+)- DMCPA has attracted much attention. In order to improve the activity and stability of Novozyme 435 for enzymatic preparation of S-(+)-DMCPA from 2,2-dimethylcyclopropane carboxylate (DMCPE), the glutaraldehyde modification for Novozyme 435 was investigated and the glutaraldehydemodified Novozyme 435 was used as biocatalyst for the synthesis of S-(+)-DMCPA. The results showed that the modified Novozyme 435 had a better reusing merit than unmodified enzyme. The maximum specific activity was obtained by modification Novozyme 435 with 1.5% glutaraldehyde solution under the conditions of shaking at 200 rpm and $30^{circ}C$ for 45 min. The optimal enzymatic hydrolysis conditions for glutaraldehyde-modified Novozyme 435 were also confirmed. The optimized hydrolytic reaction mixture contained 10 mL potassium phosphate buffer (1.0 mol/L, pH 7.6), 90 mg of DMCPE and 160 mg of glutaraldehyde-modified enzyme, and the reaction was performed at $30^{circ}C$ and 200 rpm for 52 h. The reusing efficiency of modified Novozyme 435 was further evaluated. Under the optimal conditions, the modified enzyme remained 76.0% of its original yield after 10 times reuse, but the optical purity of the product kept intact; whereas the yield of unmodified enzyme reduced to 20.8% of its initial value and the ee value of product decreased a lot to 90.7% after 7 times recycle. These results showed that the modified Novozyme 435 was more cost-effective for the preparation of S-(+)-DMCPA in industrial application.