The fermentation of L-tryptophan by Escherichia coli TRJTH0709 in 30 L bioreactor was investigated. The effects of initial glucose concentration and glucose concentration in fermentation process on specific growth rate,plasmid stability,concentration of acetic acid,biomass and yield of L-tryptophan were studied. The optimal concentration of initial glucose was 20g/L. The concentration of glucose in process of L-tryptophan fermentation was controlled by glucose-limited fed-batch mode. According to the dissolved oxygen response signal,using feedback control,the feed rate in fermentation process was effectively manipulated and the specific growth rate was controlled at low level. The accumulation of acetic acid was reduced effectively and the plasmid stability was improved obviously. Biomass of 50.21 g/L dry cell weight could be obtained and the yield of L-tryptophan was 33.50 g/L. The high cell density cultivation was achieved eventually by using the strategy of feedback control of dissolved oxygen.