The invention provides a method for separating polypeptide from an imidazolium ionic liquid and belongs to the technical field of purification and separation of biomolecules. The method for separating the polypeptide from the imidazolium ionic liquid comprises the following steps of re-extracting to remove a dissociative imidazolium ionic liquid by use of a salting-out method; then thoroughly removing imidazole ions which are combined with polypeptide molecules through chemical keys by use of a reagent containing silver ions. The method for separating the polypeptide from the imidazolium ionic liquid is simple and effective, low in cost, low in energy consumption and high in product purity, and can be used for greatly enhancing the applicability of the imidazolium ionic liquid in polypeptide chemistry and the application range of the imidazolium ionic liquid.