The efficient utilization of xylose is regarded as a technical barrier to the commercial production of bulk chemicals from biomass. Due to the desirable mechanical properties of polylactic acid (PLA) depending on the isomeric composition of lactate, biotechnological production of lactate with high optical pure has been increasingly focused in recent years. The main objective of this work was to construct an engineered Escherichia coli for the optically pure l-lactate production from xylose. Six chromosomal deletions (pflB, ldhA, ackA, pta, frdA, adhE) and a chromosomal integration of l-lactate dehydrogenase-encoding gene (ldhL) from Bacillus coagulans was involved in construction of E. coli KSJ316. The recombinant strain could produce l-lactate from xylose resulting in a yield of 0.91 g/g xylose. The chemical purity of l-lactate was 95.52%, and the optical purity was greater than 99%. Moreover, three strategies, including overexpression of l-lactate dehydrogenase, intensification of xylose catabolism, and addition of additives to medium, were designed to enhance the production. The results showed that they could increase the concentration of l-lactate by 32.90, 20.13, and 233.88% relative to the control, respectively. This was the first report that adding formate not only could increase the xylose utilization but also led to the fewer by-product levels.