Amyloid-β oligomer (AβO) is widely regarded as a reliable biomarker for the early diagnosis of Alzheimer's disease (AD). In this study, a signal on-off ratiometric electrochemical immunosensor has been developed for ultrasensitive detection of AβO. To achieve the dual-signal ratiometric strategy, ultrasmall copper sulfide nanoparticle-engineered covalent organic framework hybrid nanocomposites (CuS@COFs) were utilized as excellent electrocatalysts toward hydroquinone (HQ) oxidation to produce detectable signals. Meanwhile, electroactive thionine (Thi) and Aβ antibody-modified gold nanoparticles (Thi-AuNPs-Ab bioconjugates) were designed as another electrochemical indicator. Based on these two signals, an ultrasensitive sandwich-like electrochemical immunosensor was established for AβO detection. The introduction of AβO resulted in a remarkable decline in the electrochemical signal of HQ but an increase in the signal of Thi. Under optimum conditions, the ratios between the double signals (IThi/IHQ) showed a proportional linear relationship with the AβO concentration (1 pM-1 μM) with a low detection limit of 0.4 pM (S/N = 3), and the biosensor was able to determine the content of AβO in real cerebrospinal fluid samples with satisfactory results. The ratiometric strategy proposed in our study offers a sensitive and efficient approach for early diagnosis of AD, and this work will promote the further applications of engineered COFs in electrochemical sensors.