Bilirubin is a yellow compound found in digestive fluids, produced through the breakdown of hemoglobin (Hb). It is widely used as a biological marker for diagnosing liver disorders. The presence of bilirubin in urine is generally not considered a normal physiological condition. However, several studies have shown that trace amounts of direct bilirubin are present in neonatal patients with jaundice. This study presents the development of a non-invasive amperometric electrochemical biosensor for the indirect detection of bilirubin in urine samples without employing peroxidase as an external redox mediator. Instead, Vanadium-based nanozymes were used as artificial enzyme mimics. A calibration curve demonstrated linear response to bilirubin concentrations between 1 μM and 26 μM, with a limit of detection calculated at 1.000362 nM. The applicability of this bilirubin sensor was tested in phosphate buffer. The developed biosensor is presented as a potential alternative for detecting bilirubin in urine samples, offering a high analyte selectivity, a low detection limit, and a portable, rapid, and non-invasive detection method. The incorporation of Vanadium nanozymes contributes to its lower cost, stability, and improved accessibility. These characteristics support its potential for future integration into wearable platforms, such as baby diapers.