In order to avoid the interference of natural GAD expressed by the own genome of Escherichia coli, the recombinant cellulose-binding domain glutamate decarboxylase (CBD-GAD) was firstly immobilized by regenerated amorphous cellulose (RAC), and then the RAC-CBD-GAD immobilized enzyme was isolated and used as an evaluation index to optimize the culture conditions for highly efficient expression of CBD-GAD in E. coli GDMCC60445 by one-factor-at-a-time and Taguchi methods. The results indicated that the optimal medium for E. coli GDMCC60445 to express CBD-GAD was modified Luria-Bertani (LB) medium, which was consisted of 8 g/L tryptone, 6 g/L yeast extract, 10 g/L sodium chloride, and pH 5.5. The suitable culture conditions were 37 ℃, 120 r/min and 24 h. Under the optimal conditions, the activity of RAC-CBD-GAD prepared from E. coli GDMCC60445 was (419.29±10.37) U/g, which was consistent with the predicted value of Taguchi and improved by (30.28±3.22)% as compared to the initial one.