L-isoleucine （Ile） is one of the three branched chain amino acids that are essential to human. It is one of the important amino acids to synthesize some hormones and enzymes [1]. Ile can enhance the protein integration and inhibit the protein from degradation [2, 3]. Ile also plays a very important role in human life. Ile has been widely used as addictive in biological medicine, structuring project, photochemistry, electrochemistry, food industry and cosmetics [4-6]. It is also be used as feed addictive [7] and added to functional beverage [8].In this paper, the Brevibacterium lactofermentation strain was used to produce L-isoleucine. A series of feasible approaches or strategies were carried out to achieve high product concentration, high yield and high productivity of isoleucine in the optimization of B.lactofermentation cultivation processes, including:1. The effect of dissolved oxygen and pH on L-isoleucine production by Brevibacterium lactofermentation was investigated in batch fermentation process, a two-stage agitation speed control strategy was developed, in which the agitation speed was controlled at 700 r/min in the first 12 h to achieve a DO level of up to 35%, and then switched to 600 r/min after 12 h to maintain a DO level at 15–20%. A relative high productivity（0.094 g/g） and glucose consumption velocity(4.76 g/L^-1·h^-1) were gained, the high isoleucine production（23.3 g/L） was achieved in a relative short time（56 h）, which was increased by 11.6% compared to the single agitation speed, and the isoleucine productivity respectively increased by 83.6%、28.7%、44.9%、35.7%. In order to make sure whether the combination of DO and pH control can boost the production by a mutual effect, different control modes were conducted, based on the data obtained from the two-stage agitation speed control strategy and the analysis of kinetics parameters at different pH values. The results showed that the mode of combining two-stage DO with constant pH7.2 control strategy was the optimal, the isoleucine production reached 23.9 g/L.2. Under different initial concentrations of glucose, the cell growth and the isoleucine production were difference: the higher the initial concentration of glucose （140 g/L） may eventually enhance the duration of cell growth, but isoleucine reduced the yield of glucose （0.131 g/g）; the lower the initial concentration of glucose （100 g/L） can improve the isoleucine yield （0.148 g / L）; through fed-batch fermentation with an appropriate complement of glucose （60 g/L） maybe improve cell concentration （OD562 was 1.273）, and increase isoleucine production （23 g/L）. However, too high and too low amount of glucose were not good for isoleucine synthesis. A constant flow rate can effectively control bacterial concentration to avoid excessive cell growth so that eventually improve isoleucine production （24.5 g/L） and higher yield （0.154 g/g）. On the basis of the above analysis, a constant flow strategy was carried out: when the residual glucose concentration was less than 15 g/L, the constant flow rate of 3.3 g?L^-1?h^-1 was controlled to the end of fermentation. 3. The effect of temperature, varied from 26℃to 34℃, on production of isoleucine was investigated. Based on kinetic parameters analysis, a temperature-shifting strategy was proposed, in which, at 0-12 h, culture was performed at 31℃to obtain a high specific cell growth rate, and 12 h later, the temperature was decreased step by step from 31℃to 28℃to keep a high isoleucine production rate, and then switched temperature to 26℃until the end. A high concentration （27.7 g/L）, yield （0.78 g/g） and productivity （0.475 g/（L h）） of isoleucine were achieved by applying this strategy, which were 11.3%、21.9% and 18.8% higher than batch fermentation with constant temperature 31℃, respectively. Studied on the isoleucine biosynthesis metabolic flux under different temperature, with too low temperature, the metabolic flux to TCA cycle would reduce while the metabolic flux to by-products would increase. With the increasing of temperature, the metabolic flux to TCA cycle will increase while the metabolic flux to by-products would decrease. But too high DO, the metabolic flux to isoleucine would be few as the metabolic flux to TCA cycle was abundant. The three-stage temperature control strategy can led more carbon flow to the EMP and TCA cycle which would increase metabolic flux to isoleucine and improve the isoleucine production （increased by 11.3 %）.