The invention discloses a preparation method and an application of a 3D chip for screening of traditional Chinese medicine components. The chip comprises a support layer, an initiator layer and a polymer brush layer, wherein the initiator layer is anchored to the support layer through metal-sulfur bond covalence; the polymer brush layer is covalently bound on the initiator layer. The 3D chip for screening of traditional Chinese medicine components can fix drug micromolecules of different structures or traditional Chinese medicine components; besides, the chip is of a 3D structure, strong anti-nonspecific monomers are introduced into the polymer layer, so that the 3D chip for screening of the traditional Chinese medicine components greatly improves the fixation quantity of drug micromolecules or traditional Chinese medicine components, notably enhances the sensing signals and solves the problem about poor specificity of micromolecular array chips.