The nitrilase from Arthrobacter aurescens CYC705 was used to catalyze the synthesis of iminodiacetic acid (IDA) in this work. The biocatalyst form, optimization of biocatalysis process and scale-up of the reaction system were investigated in detail. Several biocatalyst forms including the aminated carrier immobilized nitrilase, epoxy carrier immobilized nitrilase, sodium alginate immobilized cells, chitosan immobilized cells and free whole cells were prepared and compared. It was found that the chitosan immobilized cells showed the highest catalytic efficiency and the best stability. After optimizing the reaction system, reaction temperature, metal ion concentration, substrate concentration and addition amount of chitosan immobilized cells, the optimal reaction conditions were obtained as follows: taking 50 mmol/L pH 6.6 Na2HPO4-citric acid buffer as the reaction medium, iminodiacetonitrile (IDAN) concentration 200 mmol/L, final CoCl2 concentration in the reaction medium of 1 mmol/L, reaction temperature 37 ˚C, chitosan immobilized cells addition amount 0.25 g/5 mL reaction volume. Under these conditions, IDAN could be hydrolyzed to IDA completely in 2 h. Further, the reaction system was amplified 10 times. It was found that the time required to catalyze the full conversion of 200 mmol/L IDAN to IDA was only 1h.