The invention provides a method for separating and purifying osteopontin (OPN) from human milk. The method comprises: separating OPN-containing whey from human milk samples through high-speed centrifugation; adopting high-performance liquid chromatography together with SCX chromatographic column to perform preliminary gradient separation; obtaining SCX5 and SCX6 components containing OPN; furtherpurifying the two components on C4 chromatographic column; and obtaining high-purity osteopontin OPN. The method has the advantages of simple steps, high speed, time conservation, few types of used reagents and low cost.