Abstract: Cellulose-tris(3,5-dimethylphenylcarbamate) was prepared according to a reported method, and the structure was confirmed by infrared spectroscopy and elemental Analysis. It was coated onto an aminopropylated mesopore spherical silica gel, and the final product was used as a chiral stationary phase of high performance liquid chromatography for the enantioseparation of 2-(1-hydroxyethyl)-1H-benzimidazole, which is effective against Candida tropicalis with contact action. The first enantioseparation of this compound has been achieved. Mixtures of hexane and 2-propanol were used as mobile phases. The effects of 2-propanol concentration in the mobile phase on the retention and resolution were investigated. While the concentration of 2-propanol in mobile phase was decreasing, the resolution factor was increased. When the 2-propanol concentration comes down to 10%, the resolution factor is 2.23.