Sequential optimization strategy was achieved based on statistical experimental designs for enhancement of Aspergillus awamori EM66 exochitinase production. Firstly, 2-level Plackett-Burman design was accomplished for distinguishing between the bioprocess parameters, which improve the exochitinase production. Second optimization step was implemented through central composite design (CCD), for optimization variables amounts which have the highest positive effect on exochitinase production. Maximum activity (5998mU/ml) for exochitinase reported approximately 22 fold increase compared to the basal medium activity. Mortality 92%, 86.67% and 65.67% was recorded when the partial pure fungal exochitinase was applied to the diet of the greater wax moth larvae, Galleria mellonella, the cotton leaf worm, Spodoptera littoralis, and the black cutworm, Agrotis ipsilon, respectively. The results indicated to the importance of Aspergillus awamori EM66 exochitinase as an effective bio-pesticide.