L-ornithine is a basic amino acid, which shows significant value in food and medicine industries. There is a huge space for L-ornithine production with strains available for metabolic engineering, and it is urgent to develop a high-efficiency engineering strain for industrialization. Here, xylose isomerase and xylulose kinase were introduced into Corynebacterium glutamicum S9114 to establish xylose metabolism pathway, and then xylose became a substitute carbon source of glucose. In addition, the optimization and overexpression of phosphoenolpyruvate carboxylase and pentose transporter have been conducted to promote the synthesis of L-ornithine for the first time. Furthermore, though optimizing the concentration ratio of glucose and xylose (7:3), adding biotin and thiamine hydrochloride, we arrived at the highest L-ornithine yield 41.5g/L in shaking flask fermentation so far. Our results demonstrate that the combination of metabolic engineering and the optimization of fermentation process can make great potential for L-ornithine production by lignocellulose hydrolysate.