The present invention relates to a method for preparing a conjugate by linking a physiologically active polypeptide, a non-peptidyl polymer linker, and an immunoglobulin constant region via a covalent bond. More particularly, the present invention relates to a method for efficiently preparing the physiologically active polypeptide conjugate, in which a salt is used in a coupling reaction to improve the problem of low production yield during preparation of the physiologically active polypeptide conjugate. The complex of physiologically active polypeptide - non-peptidyl polymer - immunoglobulin constant region can be produced with high purity and yield by the preparation method of the present invention. Due to the method for preparing the physiologically active polypeptide conjugate, production costs can be reduced. Therefore, the method can be used to develop long-acting formulations of physiologically active polypeptides, which have improved industrial applicability and drug regulation compliance.