This work created a simple electrochemical cortisol sensing method based on ZnO nanostructures to track high-performance athletes' stress levels and recuperation times. ZnO nanorods (ZnO NRs) were deposited on the surface of a glassy carbon electrode (ZnO/GCE) using an electrodeposition technique. Subsequently, 3-aminopropyltriethoxysilane (APTES) was applied to the electrode surface in order to improve cortisol antibody (C-M ab) immobilization and stability (C-M ab/APTES/ZnO/GCE). Successful immobilization of C-M ab on APTES/ZnO/GCE and electrodeposition of ZnO NRs on GCE were confirmed by structural investigations conducted using XRD, FT-IR, and FE-SEM. Cortisol was detected using an electrochemical immunosensor (C-M ab/APTES/ZnO/GCE) by monitoring the change in DPV and CV currents following cortisol binding to C-M ab. According to electrochemical experiments, the immunosensor exhibited a steady, selective, and sensitive response when it detected cortisol at concentrations between 10−6 nM and 106 nM, with a detection limit of 2 × 10−4 nM. It was assessed if the C-M ab/APTES/ZnO/GCE combination could identify cortisol in human serum. The samples' relative standard deviation (RSD) values range from 3.63% to 4.30%, and their recovery rates range from 90.00% to 98.50%, according to the analytical study. These findings imply that the C-M ab/APTES/ZnO/GCE offers accurate evaluations for cortisol detection in human serum samples.