The invention belongs to the technical field of biomacromolecule separation and purification, and particularly relates to a separating and purifying method for Chinese wolfberry polyose. According to the method provided by the invention, novel thermo-sensitive periodic copolymers, namely polyethylene oxide-polypropylene oxide-polyethylene oxide and inorganic salt, are used for forming a two-phase aqueous system, and small molecular weight impurities such as protein, coloring matters and the like in Chinese wolfberry polyose are removed. Inorganic salt in a lower phase is removed according to a dialysis method, and ethyl alcohol is deposited to obtain a polyose finished product; in virtue of a temperature response behavior of a thermo-sensitive polymer, and through multi-step comprehensive operations such as protein precipitation through temperature reduction, PH value adjustment, heating for phase separation and the like, the thermo-sensitive polymer in an upper phase can be recovered successfully. The invention provides a novel protein removing method which is efficient, green and low in cost, the thermo-sensitive periodic copolymer is effectively recovered and reused, the defects of high price and difficult recovering of a polymer formed through a traditional polymer aqueous two phase method can be overcome, material consumption and cost are reduced greatly, and the method can be applied to industrialized production in large scale.