A novel perphenyl carbamate of beta-cyclodextrin immobilized onto aminized silica gel via multiple ureido chemical bonds was synthesized and its enantioseparation ability was evaluated as chiral stationary phase (CSP) through separation study of fluoxetine (FLU) on high performance liquid chromatography under reversed phase condition. The optimal separation condition of FLU was found and a good separation was obtained. The influences of mobile phase composition, ionic strength, pH, flow rates of mobile phase, and column temperature on retention behavior, resolution of (R)-FLU and (S)-FLU were studied. The results showed that the new CSP had good separation ability and built a strong basis for the large-scale separation of FLU.