<p>Breast tumor–derived exosomes promise to guide diagnosis and therapy. But the small size and low mass of exosomes have higher requirements for target binding and signal amplification. Although hybridization chain reaction (HCR) has been extensively utilized for the sensitive detection of an enormous range of biomolecules, including the exosomes, achieving sufficient amplification efficiency remains challenging. To address this limitation, more and more researchers integrate the reaction system with functional materials of DNA. Inspired by this, we herein report for the first time the cubic DNA nanostructure enhanced hybridization chain reaction (CDN-HCR) to detect breast tumor–derived exosomes. Unlike other nanostructure-assisted HCR approaches, the four cholesterol anchors on the CDN enhance the binding affinity of signal probes for exosomes, while the four triggers combined with the supporting CDN further improve the reaction efficiency of HCR. Furthermore, incorporating an electrochemical detection system, the assay platform enables enzyme-free and label-free detection of exosomes. The quantitative detection of exosomes is easily achieved with a linear range of 5.5 × 10² to 5.5 × 10⁷ particles/μL and a minimal detectable concentration of 51 particles/μL. Finally, the human serum study proves the feasibility of this technique in clinic applications.</p> Graphical Abstract <p></p>