The invention relates to a nucleic acid chip and a preparation method and application thereof, belonging to the detection field of enzymes, microorganisms or nucleic acid molecules. The nucleic acid chip comprises a substrate, nucleic acid molecules are attached to a part of the surface of the substrate; a region of the substrate without the nucleic acid molecules is covered with a material capable of inhibiting cell growth; the material can be a non-biocompatible material, such as poly-N-isopropylacrylamide, polyvinyl caprolactam, polyacrylic acid, chitosan, sodium alginate, and the like or be DNA, siRNA, esiRNA, plasmid, and the like. The nucleic acid chip is particularly suitable for researching functional gene screening aspects, such as cell proliferation, cell differentiation, apoptosis, cell migration, and the like.