A method for preparing a conjugate of a physiologically active polypeptide and a non-peptide polymer by linking physiologically active polypeptide with non-peptide polymer through a covalent bond using an organic solvent is provided. A method for preparing a physiologically active polypeptide complex by linking the conjugate with a carrier is provided. The complex shows improved in vivo duration and stability of the physiologically active polypeptide. The method can prepare the conjugate at a lower production cost, and the resulting conjugate shows an extension of in vivo activity at a relatively high level and significantly increase in the blood half-life.