This study focused on the pretreatment with deep eutectic solvent of choline chloride (ChCl)/urea mixtures on rice straw and its chemical fractions of holocellulose, α-cellulose, and acid-insoluble-lignin (AIL). The pretreatment of ChCl/urea was significantly affected by the treated temperature prior to the treated time, and 130°C and 4h was an optimum condition for ChCl/urea pretreatment. The separation capacity of ChCl/urea on the chemical fractions was in an order of AIL (22.87%)> hemicellulose and amorphous cellulose (16.71%)>α-cellulose (9.60%). ChCl/urea had a higher selective solubility on lignin. The solubility of the whole fractionation of rice straw affected by ChCl/urea was a combination of solubilization on cellulose, hemicellulose and lignin. ChCl/urea pretreatment increased crystallinity index (CrI) of rice straw residue and α-cellulose, while had no obvious influence on CrI of holocellulose. The effect of structural properties of rice straw residue on enzymatic hydrolysis was also explored.