The invention discloses a preparation method and application of microspheres for purifying mRNA (messenger Ribonucleic Acid), and the preparation method of the microspheres for purifying mRNA comprises the following steps: S1) washing polystyrene microspheres with active groups on the surface by using a coupling buffer solution; s2) Oligo dT is added to react with the polystyrene microspheres, and after the reaction is finished, a coupling buffer solution is used for cleaning; and S3) adding a blocking reagent for reaction, and after the reaction is finished, cleaning with a coupling buffer solution to prepare the microspheres for purifying mRNA. The surface of the rigid polystyrene microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the rigid polystyrene microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the microsphere, so that the microsphere has mRNA separation and purification capabilities; and the microspheres have rigid structures, so that the microspheres can bear a certain pressure, and the Oligo dT microspheres are loaded into a chromatographic column, so that mRNA can be rapidly separated and purified on equipment such as liquid chromatography and the like.