The effects of pH on the fermentation of L-isoleucine were studied with L-isoleucine producing strain E.coli TRFC.The effects of pH in fermentation process on enzyme activity,biomass,yield of L-isoleucine,glucose consumption rate and accumulation of byproduct were studied by carrying out fed-batch fermentation.Results showed that the yield of biomass and L-isoleucine by three stage pH control strategy was increased by 6.1% and 9.2% compared to that from the condition of constant pH 7.2.Besides,the concentration of acetate and ammonium was decreased by 7.8% and 11.6%.The combined neutralizing agents,NaOH and ammonia,which were used to maintain pH level could effectively reduce the inhibitory effects of ammonium and delay cell aging.