The invention discloses a preparation and purification method of rice bran phenolic substances with high biological activity. The preparation and purification method comprises the following steps: firstly, crushing defatted rice bran into particles, adding an aqueous solution of ethanol, sealing and leaching a container mouth part, then performing shaking out and leaching at room temperature, centrifuging and collecting a supernatant leaching solution; secondly, performing vacuum concentration on the leaching solution until no ethanol exists, and diluting a concentrated solution by using distilled water; thirdly, performing wet column packing by selecting pretreated HPD722 macroporous resin, and sampling the diluted leaching solution of the rice bran phenolic substances; fourthly, washing with distilled water to remove impurities; when an eluate has no turbidity, starting eluting by using the aqueous solution of ethanol as an eluent; fifthly, performing vacuum concentration on the eluent until no ethanol exists, and performing vacuum freeze drying to obtain a purified extract of the rice bran phenolic substances. According to the method provided by the invention, the rice bran phenolic substances with the high biological activity can be prepared by separation.