Covalent organic frameworks (COFs) with uniform porosity, good stability, and desired biocompatibility can function as carriers of immobilized enzymes. However, the obstructed pores or partially obstructed pores have hindered their applicability after loading enzymes. In this study, the hierarchical COFs were prepared as an ideal support to immobilize glucose oxidase (GOD) and obtain GOD@COF. The hierarchical porosity and porous structures of COFs provided sufficient sites to immobilize GOD and increased the rate of diffusion of substrate and product. Moreover, N,Fe-doped carbon dots (N,Fe-CDs) with peroxidase-like activity were introduced to combine with GOD@COF to construct an enzyme-mediated cascade reaction, which is the basis of the sensor GOD@COF/N,Fe-CDs. The sensor has been successfully built and applied to detect glucose. The limit of detection was 0.59 μM for determining glucose with the proposed fluorescence sensor. The practicability was illustrated by detecting glucose in human serum and saliva samples with satisfactory recoveries. The proposed sensor provided a novel strategy that introduced COF-immobilized enzymes for cascade reactions in biosensing and clinical diagnosis. Graphical abstract