In order to meet the demand of analyzing low concentrations of DNA with high sensitivity, herein, a magnetically separated-type fluorescent biosensor has been constructed for ultrasensitive DNA detection based on a chemically driven redox-cycling system. In this study, the substrate o-phenylenediamine dihydrochloride (OPD) is oxidized to produce 2,3-diaminophenazine (DAP) with strong fluorescence emission at 555 nm. In the chemically driven redox-cycling system, Cu²⁺ oxidized OPD to yield DAP and Cu⁺/Cu⁰. On the one hand, the produced Cu⁰ could further oxidize OPD to form DAP, while on the other hand, the generated Cu⁺ could react with newly imported H₂O₂ to undergo a Fenton-like reaction to form hydroxyl radicals (˙OH) and a Cu²⁺ ion. The reproduced Cu²⁺ ion and generated ˙OH could in turn oxidize OPD to form more DAP, touching off a chemically driven chemical redox-cycling amplification reaction. By leveraging the magnetic separation mode, the chemically driven redox-cycling strategy allowed quantitative detection of H1N1, exhibiting a linear range of 1 pM to 1 nM with a low limit of detection of 300 fM. The magnetic separation mode could effectively minimize the background signal to obtain greatly improved sensitivity. It is promising that the chemically driven redox-cycling strategy could provide a novel approach in the fields of biosensing and biological sample assay.