<p>The simultaneous electrochemical determination of multiple biologically active molecules remains one of the greatest challenges in modern analytical chemistry because of their structural similarities, closely overlapping oxidation potentials, and strong mutual interferences. The present study focuses on a novel electrochemical sensing platform that enables the simultaneous, precise, and interference-free determination of 4-aminophenol (4-AP), dopamine (DA), and acetaminophen (AC) using a strategically engineered nanocomposite-modified electrode. The key innovation lies in the rational surface modification, typically involving noble metal nanoparticles (AuNPs), metal oxide (CuO-NiO) nanostructures, and carbon-based nanomaterials (SWCNTs), which enhance electron transfer kinetics and spatial separation of the oxidation peaks, thereby minimizing cross-reactivity. The desired nanocomposite was fabricated using a simple two-step synthesis route and characterized with several spectroscopic and microscopic techniques. The sensor electrode exhibits outstanding electrocatalytic performance due to its larger surface area, lower overpotential, and reduced <i>R</i>_ct value, facilitating electron transfer during the sensing event. Differential pulse voltammetry (DPV) analysis yielded sensitivities of 1.374 µA/(µM·cm²) for 4-AP, 1.203 µA/(µM·cm²) for DA, and 0.129 µA/(µM·cm²) for AC. The limits of detection (LODs) were determined to be 0.106 µM, 0.122 µM, and 1.13 µM for 4-AP, DA, and AC, respectively. The proposed sensor demonstrates excellent selectivity for target biomolecules even in the presence of various interfering species. Additionally, the sensor exhibits outstanding stability under the current experimental conditions. Finally, recovery analysis was performed with two different ages of human blood serum (ages 27 and 67) to evaluate the sensor’s efficiency for real-life applications.</p>