Through the biorefinery process, xylose can be derived from lignocellulosic biomass waste, such as sugarcane trash (ST). Acid hydrolysis of biomass is one of the potential methods to be applied for producing xylose. This study aimed to optimize the stepwise and repeated use of hydrolysate in the acid hydrolysis of ST with the assistance of an autoclave and microwave digester for xylose production, which was further used to produce xylitol. Step 1 involved hydrolysis with oxalic acid in an autoclave, while Step 2 used hydrolysate from Step 1 in a microwave digester with a new ST feedstock. Meyerozyma caribbica InaCC Y67 was used to examine the fermentation of hemicellulose hydrolysate obtained under ideal hydrolysis conditions to produce xylitol. The results show that the optimum conditions for xylose production are a S/L ratio of 1:10, 2% oxalic acid, and 60 min of heating in Step 1, followed by 7.5 min at 180 °C using the hydrolysate from Step 1. The repeated use of hydrolysate may increase the concentration of xylose in the hydrolysate. The final xylose concentration obtained in Step 2 hydrolysis (35.2 g/L) is approximately twice that of Step 1 (14.6 g/L). The yeast M. caribbica InaCC Y67 successfully produced xylitol (6.49 g/L) regardless of the presence of high concentrations of inhibitors in this non-detoxified hydrolysate. These results show that ST can be a potential source of xylose for xylitol synthesis through a stepwise hydrolysis and repeated use of hydrolysate.