As　a　kind　of　cell-free　DNA　in　the　bloodstream　liberated　from　tumor　cells,　circulating　tumor　DNAs　(ctDNAs)　have　been　recognized　as　promising　biomarkers　in　the　field　of　early　cancer　diagnosis.　However,　robust,　sensitive,　and　accurate　detection　of　ctDNA　in　serum　remains　extremely　challenging,　especially　toward　the　mutant　KRAS　gene,　one　of　the　most　frequently　mutated　genes.　Although　DNA　oligonucleotides　as　emerging　practical　signaling　materials　have　been　developed　as　sensitive　and　accurate　tools,　some　intrinsic　defects　need　to　be　overcome,　such　as　fragility　in　complex　biological　environments.　In　this　work,　on　the　basis　of　the　hydrophilicity-promoted　assembly,　a　core/shell　DNA　nanostructure　(DNS-MB)　probe　is　constructed　from　only　one　hairpin-shaped　probe　(cholesterol-modified　palindromic　molecular　beacon,　Chol-PMB)　for　the　amplification　detection　of　KRAS　mutation　in　serum　without　the　need　for　any　auxiliary　probe.　Chol-PMB　is　designed　to　recognize　target　DNA　and　serve　as　a　polymerization　primer　and　template,　and　thus　target　species　can　initiate　polymerization-based　strand　displacement　amplification　(SDA).　Moreover,　target　DNA　is　able　to　induce　further　aggregation　of　DNS-MB　particles　due　to　the　enzymatic　cross-linking　effect,　leading　to　a　structural　upgrade.　The　DNS-MB　probe　exhibits　a　detection　limit　of　50　fM　and　a　wide　quantitative　range　(from　50　fM　to　160　nM).　In　addition,　single　nucleotide　polymorphisms　can　be　discriminated,　such　as　mutant　KRAS　G12D　(KRAS-M),　providing　a　desirable　platform　for　screening　ctDNAs.　More　excitingly,　because　the　termini　of　DNA　components　are　hidden　inward　from　nuclease　attack,　DNS-MB　circumvents　a　false-positive　signal　even　in　freshly　sampled　serum　and　is　suitable　for　application　in　the　complex　biological　milieu.　As　a　proof　of　concept,　the　DNS-MB　probe　is　expected　to　provide　useful　insight　into　the　development　of　simple　and　degradation-resistant　DNA　probes　for　substantially　amplified　detection　of　ctDNAs　in　complex　serum,　showing　potential　applications　in　the　field　of　early　tumor　diagnosis.　©　2021　American　Chemical　Society.