<p>An ultra-sensitive fluorescent biosensor based on CDs-Eu@ZIF-8 Modified Fe₃O₄@AuNPs Magnetic Nanoparticles was constructed for sensitive detection of DENV-2 Nucleocapsid Protein 1 (NS1). Briefly, we doped Eu³⁺ into carbon dots using a new synthesis method, preparing Eu³⁺-doped carbon dots with novel excitation and emission wavelengths (CDs-Eu). Compared with CDs, CDs-Eu exhibits superior fluorescence properties. The fluorescence material was encapsulated in zeolitic imidazolate frameworks (ZIF-8) to form stable and signal amplification nanoparticles CDs-Eu@ZIF-8. To achieve highly sensitive detection of NS1, we fabricated multilayered fluorescent nanocomposites of Fe₃O₄@AuNPs-DNA3-CDs-Eu@ZIF-8. Additionally, Ab1 was immobilized on protein A/G magnetic beads (A/G MB) for target capture, and the other antibody (Ab2) was conjugated to the DNA1/DNA2 duplex to form target detection probes. In the presence of NS1 protein, the Ab1 immobilized on A/G MB and Ab2 composite (Ab2-DNA1/DNA2) simultaneously recognized the target NS1, and after magnetic separation, EXO III was introduced to hydrolyze the DNA1 strands, resulting in the release of the DNA2 strands. The liberated DNA2 strands hybridize with the complementary DNA3 strands immobilized on Fe₃O₄@AuNPs-DNA3-CDs-Eu@ZIF-8 nanoparticles, forming DNA2/DNA3 duplexes. This hybridization triggers the dissociation of the CDs-Eu@ZIF-8 nanocomposites from the nanoparticle surface. Furthermore, EXO III degrades the DNA3 strands in the duplex, leading to the release of DNA2, which acts as a catalytic DNA walker to initiate cyclic hybridization and degradation events, thereby amplifying the fluorescence signal. The platform demonstrates high sensitivity and a broad dynamic range for NS1 detection, achieving a linear response from 0.02 ng/mL to 10³ ng/mL with a detection limit of 18.2 pg/mL.</p> Graphical abstract <p></p>