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Research methods for DNA detection have been widely extended since the application of nanotechnology, but it remains a challenge to detect specific DNA sequences or low abundance genes in biological samples with accuracy and sensitivity. Herein, a novel biosensor is proposed for the analysis of target DNA in maize based on a high-density "hot spot" surface-enhanced Raman scattering (SERS) substrate and hybridization chain reaction (HCR) amplification strategy. Finite-difference time-domain (FDTD) simulation confirmed that the electromagnetic field enhancement at the gaps between the adjacent Au nanoflowers (AuNFs) was much richer than that around a single AuNF, indicating that the aggregated AuNFs had a good SERS signal amplification effect. Based on the high-density "hot spots" AuNF substrate, the HCR of biotin-hairpin DNA (bio-H1 and bio-H2) could be triggered by the existence of target DNA and a long double-stranded DNA (dsDNA) could be formed rapidly. Due to the biotin and streptavidin (SA) interaction, SERS probes prepared by modifying biotin-DNA-cyanine 5 (bio-DNA-Cy5) on the surface of Ag–Au core–shell nanorods (Ag–AuNRs) could be assembled along the dsDNA, causing significant signal enhancement. The developed biosensor displayed a significant sensitivity with a broad detection range covering from 0.01 to 1 × 104 fM, and the limit of detection (LOD) was as low as 6.31 aM. Moreover, SERS was applied to detect target DNA in maize extract to verify the feasibility of the detection strategy. This strategy could not only carry out the ultrasensitive detection of target DNA but also provide an informative supplement to the SERS biosensing platform. [ABSTRACT FROM AUTHOR]