The invention discloses a multivalent aptamer functionalized DNA nanostructure probe as well as a preparation method and application thereof, one side of the DNA nanostructure of the probe is provided with a plurality of DNA aptamers, and the DNA aptamers can be specifically combined with a circulating tumor cell membrane surface protein receptor; a plurality of biotin sites are arranged on the other side of the substrate and can be combined with streptavidin modified magnetic particles; the probe is synthesized by six DNA single chains, namely S1, S2, N1, N2, N3 and SYL3C, and DNA sequences of the S1, the S2, the N1, the N2, the N3 and the SYL3C are respectively shown as SEQ ID NO.1-6. The probe disclosed by the invention can realize target cell recognition based on different expression levels of biomarkers on the surface of a cell membrane, the probe disclosed by the invention is simple to synthesize, high in CTCs capturing efficiency and easy to operate, and meanwhile, the enriched CTCs can be released through DNA hydrolase, so that the probe has potential significance on pathological diagnosis and other subsequent researches of the CTCs.