An ultrasensitive split-type fluorescent immunobiosensor has been reported based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. In this strategy, Cu2+ could oxidize chemically o-phenylenediamine (OPD) to generate photosensitive 2, 3-diaminophenazine (DAP) and Cu+/Cu0. On one hand, the generated Cu0 in turn catalyzed the oxidation of OPD. On the other hand, the introduced H2O2 reacted with Cu + ion to produce hydroxyl radicals (·OH) and Cu2+ ion through a Cu + -mediated Fenton-like reaction. The produced ·OH and recycled Cu2+ ion could take turns oxidizing OPD to generate more photoactive DAP, which triggering a self-sustaining chemical redox-cycling reaction and leading to a remarkable fluorescent improvement. It was worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Based on the H2O2-triggered cascade signal amplification, the strategy was exploited for the construction of split-type fluorescent immunoassay by taking interleukin-6 (IL-6) as the model target. It was realized for the ultrasensitive determination of IL-6 in a linear ranging from 20 fg/mL to 10 pg/mL with a limit of detection of 5 fg/mL. The study validated the practicability of the cascade signal amplification on the fluorescent bioanalysis and the superior performance in fluorescent immunoassay. It is expected that the strategy would offer new opportunities to develop ultrasensitive fluorescent methods for biosensor and bioanalysis. An ultrasensitive split-type fluorescent immunobiosensor has been reported for IL-6 detection based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. Display omitted •An ultrasensitive fluorescent immunoassay was realized by signal amplification.•The split-type immunoassay dramatically minimized the background interferences.•The sensitivity was improved by over 2-3 orders of magnitude than that of ELISA.•The strategy was coupled with chemical redox-cycling and Fenton-like reaction.