Simultaneous bioconversion of xylose and glycerol to xylonic acid and 1,3-dihydroxyacetone (DHA) was realized by using Gluconobacter oxydans (G. oxydans). Currently, the enzymatic hydrolysate to ethanol-fermented waste liquid and the inorganic acid pre-hydrolysate that contain abundant glycerol and xylose were difficult to be utilized or disposed. Based on the method of compressed oxygen supply-sealed and stirred tank reactor system (COS-SSTR), the xylonic acid and 1,3-dihydroxyacetone could be co-produced rapidly with the mixture of the dilute sulfuric acid pre-hydrolysate and ethanol-fermented waste liquid of enzymatic hydrolysate (MPEW) as material. By means of the system, we finally produced 102.3 +/- 3.2 g/L xylonic acid and 40.6 +/- 1.8 g/L 1,3-dihydroxyacetone at yield of 92.4 +/- 2.8 % and 80.6 +/- 3.5 % directly and simultaneously from the mixed solution. The central features of this bioprocess application would enable cost-competitive bacterial xylonic acid and 1,3-dihydroxyacetone production from lignocellulosic materials.