Pre-treatment system of cellulose biomass was investigated for the efficient production of bioethanol by enzymatic hydrolysis. A system of consecutive heating and high-pressure fluid milling was developed in which a part of the upstream piping of the mill was heated, which enables the cellulose biomass to be decomposed in single pass. Two cellulose biomasses, Japanese cedar with lignin and crystalline cellulose (CEOLUS TG (Asahi Chemical Industry Co., Ltd.)) without lignin, were treated by the system. When heated at 453 K (180 degrees C), both samples were decomposed and fibrous particles were observed by SEM microscopy. Using cellulase and hemicellulase, enzymatic hydrolysis of the hydrothermally treated samples was demonstrated. When hydrolysis was performed using CEOLUS treated at various temperatures, high enzymatic degradation ratio was obtained under all heating conditions. Treatment at more than 423 K (150 degrees C) gave a higher degradation ratio. When Japanese cedar samples were treated at 453 K for 110 h, holocellulose degradation ratio was two times higher than that for room temperature treatment, and alpha-cellulose degradation ratio was approximately three times higher.