Nuclease-resistant　assay　probes　are　of　significant　importance　for　biochemical　analysis　and　disease　diagnosis.　In　this　contribution,　a　reconfigurable　lipidic　moiety-attached　DNA　nanoparticle　(LDN)　is　constructed　from　a　cholesterol-conjugated　multifunctional　hairpin-type　DNA　probe　(Chol-DP)　by　hydrophobicity-mediated　self-assembly.　The　LDN　holds　high　serum　stability　and　displays　a　low　false-positive　signal　even　in　a　complex　biological　milieu.　The　hydrophobic　cholesterol　moiety　enables　the　hydrophobicity-mediated　assembly,　while　hydrophilic　DNA　sequence　serves　as　a　recognition　element　and　a　polymerization　template.　The　initiator-activated　strand　displacement　amplification　(SDA)　reaction　can　convert　the　hairpin-shaped　probe　into　rigid　double-stranded　DNA　(dsDNA),　causing　the　conformational　rearrangement-based　LDN　swelling　that　can　be　used　to　reliably　and　fluorescently　signal　the　cancer-related　p53　gene.　The　size　increase　and　structural　reconfiguration　are　confirmed　by　dynamic　light　scattering　(DLS)　analysis　and　confocal　microscopy　imaging,　respectively.　Target　p53　is　specifically　detected　down　to　10　pM.　The　whole　assay　process　involved　only　several　simple　mixing　steps.　Recovery　test　and　blind　test　further　confirm　the　feasibility　of　the　use　of　the　LDN　for　the　detection　of　target　DNA　in　a　complex　biological　milieu,　indicating　a　promising　nanotool　for　biomedical　applications.　©　2022　American　Chemical　Society