As an essential amino acid, L-isoleucine can improve endurance and assists in the repair and rebuilding of muscle and serve as a major supplement in infusions and special diets. So far, Corynebacterium glutamicum is commonly used for the industrial production of L-isoleucine, however, period of L-isoleucine fermentation by the bacterium is relative long. In this study, we aimed at constructing a genetically defined E. coli strain capable of producing L-isoleucine using L-threonine-producing THRD as a starting strain. It was modified by co-overexpressing desensitized threonine dehydratase (encoded by ilvA) and acetohydroxy acid synthase III (encoded by ilvIH) to further increase the flux from L-threonine to L-isoleucine. The final engineered strain was able to produce 1.54 g/L of L-isoleucine by shake flask fermentation. The design principles described in this study would be useful to construct strains for producing other similar biological products.