The invention discloses a refining method for pitocin [4-Glu, 5-Asp] impurities. The refining method comprises the step of adopting high performance liquid chromatography for performing reverse enrichment, reverse salt conversion and reverse purification on a pitocin [4-Glu, 5-Asp] impurity crude product solution in turn; the pitocin [4-Glu, 5-Asp] impurity crude product solution is acquired by oxidizing a solid-phase synthesized reduction type pitocin [4-Glu, 5-Asp] impurity crude product solution; the filler of the high performance liquid chromatography is super water-tolerant; an online reverse enrichment, reverse salt conversion and reverse purification one-step process is adopted for preparing a purified polypeptide product; removal rate of impurities in the crude product solution ishigher; the production technology is optimized; an aqueous solution is served as a mobile phase in the stages of column balance, sample enrichment and salt conversion, and is environment-friendly andpollution-free; the discharged effluent can be directly subjected to sewage treatment and then can be reused.