considered to be a promising pretreatment technology up to the present [2]. The feedstock was generally catalyzed with dilute H2SO4 at high temperature. The hemicelluloses fraction was dissolved into aqueous solution and then generated a liquid as the acid hydrolysis hydrolysate. Once pretreatment is completed, the resulting cellulose fraction in solid residues was further hydrolyzed to glucose with cellulase. Afterward, the hydrolysates were fermented by suitable microorganisms and converted into ethanol. Pichia stipitis is a nature microorganism can ferment brand sugar including glucose and xylose into ethanol with higher efficiency. To evaluate the feasibility of cofermentation with Pichia stipitis was needed. Until now, the extensively studies on real hydrolysate fermentation with P.stipitis were still lacking. In the present works, we use rice straw hydrolysates to evaluate the fermentability by P.stipitis and investigated the feasibility of cofermentation process employed. The potential degradation compounds in the hydrolysate were also identified and evaluated the negative effect on ethanol fermentation.