The isomers separation process of high concentration paeoniflorin and albiflorin by three-zones SMB.was described. The working parameters of SMB were set as follows. Mode: 1-1-2; injection concentration: 40 mg/mL; flow rate of injection : 0.3mL/min; flow rate of elution: 2mL/min; flow rate of extract: 4mL/min; switch time: 25min; mobile phase: methanol∶water(V/V)=30∶70. The paeoniflorin concentration in extraction is 92% by standard curve method, and recovery is 85%; the albiflorin concentration in raffinate is 94%, and recovery is 87%; Simultaneously, the main factors which affect the separation process in SMBC were discussed.