Zizyphi Spinosi Semen is well known as a kind of herb for the treatment of insomnia and anxiety. However, due to the lack of purified compounds for functional tests, limited data proves pharmacological activities of each jujubasaponin monomer. In this study, we developed a novel method for the preparative separation and purification of jujuboside A, A(1), B, and B-1, simultaneously, from Zizyphi Spinosi Semen. Macroporous resin and reversed-phase flash chromatography was successfully used and gave a good resolution of each jujubasaponins monomer. Jujuboside A, A(1), B, and B-1 with a purity of over 98% were yielded.