<p>The increasing demand for objective training-load assessment has accelerated the development of biomarker-based monitoring strategies. Here, we present an original research study describing a non-invasive dual-plex electrochemical immunosensor for simultaneous quantification of salivary cortisol and α-amylase, integrating two key stress pathways: the hypothalamic–pituitary–adrenal and sympathetic-adreno-medullary axes. The sensor was constructed on a dual-channel screen-printed electrode modified with a ZIF-8/rGO/AuNPs hybrid nanocomposite, providing high conductivity and antibody immobilization capacity. Electrochemical impedance spectroscopy confirmed the sequential fabrication steps, with the charge-transfer resistance decreasing from 550&#xa0;Ω (bare electrode) to 180&#xa0;Ω after nanocomposite modification and increasing to 2150&#xa0;Ω upon analyte binding. Under optimized conditions (20&#xa0;min incubation, pH 7.4), differential pulse voltammetry revealed a linear response for cortisol from 1.0&#xa0;pg/mL to 100&#xa0;ng/mL (<i>R</i>² = 0.996, LOD = 0.35&#xa0;pg/mL) and for α-amylase from 10 to 2000 U/mL (<i>R</i>² = 0.992, <i>LOD</i> = 3.5&#xa0;U/mL). Selectivity tests showed less than 5% interference from selected salivary components and no measurable cross-reactivity between channels. The device exhibited high reproducibility (RSD &lt; 5%), repeatability (RSD &lt; 4%), and stability (&gt; 90% signal retention after 4&#xa0;weeks). In spiked simulated saliva, recovery ranged from 96.8 to 104.2% for cortisol and 95.5% to 103.7% for α-amylase, with excellent agreement with commercial-assay kits (<i>R</i>² &gt; 0.98). Mixed-analyte simulated-saliva tests further supported dual-channel operation in a shared sample. These results demonstrate the sensor’s analytical sensitivity and robustness in a controlled saliva-like matrix and support its further development toward practical salivary stress-biomarker monitoring after validation in real human saliva.</p> Graphical abstract <p></p>