Abstract: In order to improve the efficiency of preparing antioxidant peptides of Pinctada fucata by recombinant E.coil( DE3-pEGX6p-1-PFMAP),fermentation factors including induction temperature,induction time,concentration of IPTG,culture medium p H,shaker speed and inoculum size were optimized respectively in shake flask.Furthermore,the orthogonal experiment for the pilot-scale was optimized. Results showed that the best fermentation conditions of both shaking flask and orthogonal experiment,including induction temperature,induction time,concentration of IPTG,culture medium pH,shaker speed and inoculum size were 30 ℃,4 h,0.2 mmol / L,p H7,220 r / min,5%( v / v) respectively.And the process in this study was easy to operate and take a shorter time,which help further industrial application for preparing the antioxidant peptides of Pinctada fucata by recombinant E.coil.