Abstract: G4-amylase catalyses hydrolysis of 1,4-α-D-glucosidic linkages in amylaceous polysaccharides to remove successive maltotetraose residues from the non-reducing chain ends. As a new type of exo-amylase,it is widely used in food,medical care and other fields. The gene sequence of this enzyme from Pseudomonas saccharophila was optimized,and the gene was synthesized and then cloned into expression vector pWB980. The protein with the activity of G4-amylase has been detected in the culture medium of Bacillus subtilis pWB980-G4 /1A747. Hydrolysis product analysis of seven sources of starch showed that only maltotetraose were presented. By DNS determination,the optimum temperature was 50 ℃,the optimum pH was 7. 0. The further research indicated that the optimal fermentation conditions were activation time 5 h,inoculum 5%( vol),loading liquid amount 20 mL in 150 mL,culture time 24 h and culture temperature 37 ℃.