In this paper, we present details of the development of a disposable, noninvasive electrochemical aptasensor for the real-time monitoring of cortisol in human sweat. The sensor is fabricated on a flexible screen-printed carbon electrode (SPCE) modified with a novel conjugate of a cortisol-specific DNA aptamer and gold nanozymes (Au-NZs; gold nanoparticles that display enzyme-mimetic catalytic activity). This synergistic design leverages the aptamer’s specificity and the peroxidase-like/oxidase-like activity reported for Au nanozymes, providing a stable, enzyme-free catalytic interface that supports robust electrochemical transduction and, where applicable, catalytic amplification. Electrochemical characterization using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) demonstrated superior conductivity and a clear detection mechanism based on cortisol binding. The sensor exhibited excellent analytical performance with a clinically relevant linear detection range of 5.0 to 150.0 ng/mL, a low limit of detection of 1.5 ng/mL, and high selectivity against physiological interferents. The sensor’s practical utility was validated through the dynamic tracking of cortisol fluctuations in human sweat during physical exercise, showing an excellent correlation (r = 0.989) with the gold-standard ELISA method. This nanozyme-based platform represents a significant advancement toward accessible, point-of-care stress monitoring for applications in personalized medicine, sports science, and occupational health.