This study focused on the optimization of xylitol production from sugarcane bagasse by using response surface methodology (RSM). Xylitol was produced through a series of processes, firstly, optimization of ultrasound assisted mild alkaline pretreatment for the xylan extraction from sugarcane bagasse followed by enzymatic hydrolysis of xylan to xylose by enzyme β-1,4-xylanase and finally microbial fermentation of xylose to xylitol using yeast (Candida guilliermondii), bacteria (Corynebacterium glutamicum) and their mixed culture for different time periods (0-96 h). Maximum xylan recovery of 12.059% (w/w) was observed at pretreatment; 0.73 M NaOH, 1:38.55 solid to liquid ratio and 34.77 min ultrasonication. The enzyme concentration of 400 U/g xylan at 48 h of incubation showed the highest xylose production (81.51 mg/g bagasse). Yeast (Candida guilliermondii) resulted in the highest xylitol yield (Yp/s= 0.43 g/g) after 72 h. This bioprocess route can contribute as a suitable alternative for chemical methods of xylitol production.