Provided is a method of preparing a physiologically active polypeptide conjugate, in which a physiologically active polypeptide and a non-peptidyl polymer are linked to each other via a covalent bond. The method is to improve an overall yield of the physiologically active polypeptide conjugate by improving a reaction of the non-peptidyl polymer and the physiologically active polypeptide, and particularly, the method is to prepare a physiologically active polypeptide conjugate in a high yield by performing a two-step reaction through selective precipitation. The preparation method of the present invention is used to produce a non-peptidyl polymer-physiologically active polypeptide conjugate and a physiologically active polypeptide-physiologically active carrier conjugate in a high yield, and therefore, the method may be used in the development of long-acting formulations of various peptide drugs which maintain in vivo activity at a relatively high level and have remarkably increased blood half-life.
