The invention discloses a preparing method of xylitol. Xylose, glucose, xylose reductase, glucose dehydrogenase, coenzyme, CaCO3 and water form a biological catalysis system, so that xylose is catalyzed to be subjected to a reduction reaction to generate xylitol. Specifically, xylose reductase and NADPH serve as a catalysis system to catalyze xylose to be subjected to the reduction reaction to generate xylitol; glucose dehydrogenase serves as a regenerated catalyst of NADPH, glucose is catalyzed to be subjected to an oxidation reaction to generate gluconic acid, and NADP+ is catalyzed to be subjected to a reduction reaction to generate NADPH. Dual enzymes are adopted to produce xylitol, and after the reaction is conducted for 24 h, xylose and glucose are completely consumed; in products, the converting rate of xylitol is 98%, the concentration of xylitol can reach 146.23 g/L, the converting rate of gluconic acid is 95%, and the concentration of gluconic acid can reach 185.05 g/L.