A method for preparing a conjugate of insulin and a non-peptide polymer, comprising: reacting insulin having two N-terminals with non-peptide polymer in the reaction solution comprising an organic solvent to link said insulin with said non-peptide polymer, wherein the non-peptide polymer has reactive aldehyde groups on both ends; and isolating and purifying the conjugate of insulin covalently bound to non-peptide polymer from the reaction mixture of step; wherein the reaction solution comprises the organic solvent in an amount of 30 to 55% by volume based on the total volume of the reaction solution; the reaction solution has a pH of 4.5 to 6.5; the organic solvent is selected from the group consisting of isopropanol, ethanol and methanol; the non-peptide polymer is bound to N-terminal of beta chain of the insulin; and the insulin is a) native insulin, or b) a derivative that is prepared by any one of substitution, addition, deletion, modification, or combinations thereof in one or more amino acids of native insulin, having (i) amino acid sequence homology of at least 80% compared to the native insulin having the alpha chain sequence of SEQ ID NO. 1 and the beta chain sequence of SEQ ID NO. 2, and (ii) the function of controlling blood sugar in the body, or c) an insulin fragment wherein (i) one or more amino acids are added to, or deleted from, amino or carboxy terminals of insulin, and (ii) the insulin fragment retains the function of controlling blood sugar in the body. reacting insulin having two N-terminals with non-peptide polymer in the reaction solution comprising an organic solvent to link said insulin with said non-peptide polymer, wherein the non-peptide polymer has reactive aldehyde groups on both ends; and isolating and purifying the conjugate of insulin covalently bound to non-peptide polymer from the reaction mixture of step; the reaction solution comprises the organic solvent in an amount of 30 to 55% by volume based on the total volume of the reaction solution; the reaction solution has a pH of 4.5 to 6.5; the organic solvent is selected from the group consisting of isopropanol, ethanol and methanol; the non-peptide polymer is bound to N-terminal of beta chain of the insulin; and the insulin is a) native insulin, or b) a derivative that is prepared by any one of substitution, addition, deletion, modification, or combinations thereof in one or more amino acids of native insulin, having (i) amino acid sequence homology of at least 80% compared to the native insulin having the alpha chain sequence of SEQ ID NO. 1 and the beta chain sequence of SEQ ID NO. 2, and (ii) the function of controlling blood sugar in the body, or c) an insulin fragment wherein (i) one or more amino acids are added to, or deleted from, amino or carboxy terminals of insulin, and (ii) the insulin fragment retains the function of controlling blood sugar in the body. a) native insulin, or b) a derivative that is prepared by any one of substitution, addition, deletion, modification, or combinations thereof in one or more amino acids of native insulin, having (i) amino acid sequence homology of at least 80% compared to the native insulin having the alpha chain sequence of SEQ ID NO. 1 and the beta chain sequence of SEQ ID NO. 2, and (ii) the function of controlling blood sugar in the body, or c) an insulin fragment wherein (i) one or more amino acids are added to, or deleted from, amino or carboxy terminals of insulin, and (ii) the insulin fragment retains the function of controlling blood sugar in the body.