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Summary: The aim of this study was to develop an effective one‐step decoloration and deproteinisation method using macroporous resin for Cordyceps militaris polysaccharide (CMP) extraction. Performances of different resins were compared, and the structures and bioactivities of CMP recovered using this one‐step method were evaluated and compared with traditional methods. The results showed that NKA‐9 resin exhibited the highest adsorption capacity for pigment and free protein than other tested resins. Under the optimised condition, NKA‐9 resin adsorption exhibited high decoloration ratio (80.69%), deproteinisation ratio (62.51%) and polysaccharide recovery ratio (78.08%). Furthermore spectroscopic analysis revealed that the structure of CMP remained unchanged after adsorption, but some reagent residues can be detected using traditional methods. Compared with traditional methods, the antioxidant activity of CMP extracted using a one‐step method was significantly higher, without negative impact on cell viability. Thus, this novel technique is a promising alternative method for CMP separation and purification. [ABSTRACT FROM AUTHOR]