Enhancing the activity of fragile enzymes is greatly useful for various purposes, including fabrication of enzyme-based immunosensors. Herein, we report a defect-engineering strategy for encapsulating enzymes within covalent organic frameworks (COFs), enabling the resulting immobilized enzymes with excellent catalytic activity and stability to construct high performance immunosensors. In this design, by consciously introducing monoaldehyde ligands into the imine-linked COFs structure, we have precisely customized the structural defects to improve enzyme loading capacity and conformational stability. Defect-engineering interaction modulation between enzymes and COFs drives the enhancement of catalytic performance. Compared to the pristine COFs, the enzyme@defective COFs composites with optimally tuned catalytic performance exhibit a 4.49-fold enhancement in enzymatic activity. Furthermore, it is demonstrated that the stable skeletons of COFs provide exceptional protection for the enzymes against external perturbations. Thereafter, the optimized enzyme@defective COFs are employed to fabricate immunosensor. We have successfully established a detection method for prostate-specific antigen (PSA), achieving a low detection limit of 0.09 ng/mL. More importantly, the developed immunosensor has successfully distinguished the prostate cancer patients from healthy individuals. This work establishes a novel paradigm for enzyme immobilization, ultimately empowering the construction of a PSA immunosensor with high sensitivity, remarkable operational stability, and great clinical application potential.