The invention discloses a refining method of oxytocin [-NH2] impurities. The refining method comprises the following steps of sequentially performing antiphase enrichment, antiphase salt transformation and antiphase purification on an oxytocin [-NH2] impurity crude product solution through an high-efficiency liquid phase reverse-phase chromatography, wherein the oxytocin [-NH2] impurity crude product solution is obtained through oxidization of a solid-phase synthesis reduced oxytocin [-NH2] impurity crude product solution, and the filling of the high-efficiency liquid phase reverse-phase chromatography is ultra waterproof filling. According to the refining method disclosed by the invention, polypeptide pure products are prepared by an on-line antiphase enrichment, antiphase salt transformation and antiphase purification one-step method, the removal rate of impurities in oxytocin [-NH2] impurity crude products is high, and the purity of obtained oxytocin [-NH2] impurity pure products ishigh. Mobile phases in a column balancing stage, a sampling enrichment stage and a salt transformation stage are aqueous solutions, are environmentally-friendly and pollution-free, and effluent wasteliquid can be directly subjected to waste water treatment and recycling.