Klebsiella oxytoca is widely used for the biological production of 2,3-butanediol (2,3-BDO), a promising platform chemical with a broad range of applications. Here, to improve cell growth and production of 2,3-BDO under high concentration of xylose (100 g/L), we engineered K. oxytoca using an adaptive laboratory evolution and a biosensor-derived high throughput screening strategy. First, we developed a XylR-dependent xylose biosensor for the detection of intracellular xylose, and K. oxytoca containing the xylose biosensor was used for adaptive laboratory evolution in 100 g/L xylose. Cells were isolated by FACS screening, and the isolated strain (KO8S16) showed much improved cell growth with high xylose consumption rate (1.35 g/L/h) and 2,3-BDO productivity (0.53 g/L/h) compared with the wild-type strain. Through whole genome resequencing, it was revealed that a mutation in OmpR (a response regulator of osmotic stress) allowed to withstand high concentrations of xylose. Finally, fed-batch cultivation was performed by feeding high concentration of xylose, and K. oxytoca successfully produced 2,3-BDO at a concentration as high as 57.5 g/L by consuming 238.13 g/L xylose in 47 h.