In this work, we developed a novel PdNPs–P3HT modified ITO electrode that provides a highly sensitive and reliable platform for cholecalciferol detection. Comprehensive material characterization through UV-Vis, FTIR, XRD, SEM, and AFM confirmed the successful formation of the nanocomposite with an electroactive surface area of 0.2064 cm² and a crystallite size of 24.19 nm. The sensor demonstrated excellent electrochemical performance with a sensitivity of 26.16 µA·ng⁻¹·mL·cm⁻², an impressively low detection limit of 0.29 ng·mL⁻¹, and wide linear dynamic ranges of 0.05–50 ng·mL⁻¹ (DPV) and 0.1–2 µM (CV), ensuring accurate measurement across diverse concentration levels. Beyond its analytical robustness, the electrode showed high reproducibility, strong selectivity against interfering species, and remarkable stability over repeated use. Most importantly, it was capable of detecting trace levels of cholecalciferol even in tear samples, opening possibilities for non-invasive and cost-effective biomedical diagnostics. These findings highlight the promise of PdNPs–P3HT/ITO electrodes as next-generation tools for rapid, sensitive, and practical monitoring of vitamin D deficiency.