A novel biosensor for uric acid detection was developed using green-synthesized molybdenum oxide (MoO3) thin films. The MoO3 thin films were synthesized using Citrus limetta pith extract, which is typically considered waste, showcasing an innovative approach to repurposing waste materials. The fabrication process involved the deposition of a MoO3 thin film onto an indium-doped tin oxide (ITO) glass substrate via chemical solution deposition, followed by the immobilization of the uricase enzyme on the fabricated film. The fabricated sensors showed a high sensitivity of 212 µA/(mM·cm2) for uric acid, covering a wide range of concentrations within the physiological range. It also had a quick response time of 5 s and excellent selectivity against common interferents in plasma. The low Michaelis–Menten constant (Km) of 0.0537 mM indicates that the immobilized uricase enzyme has an enhanced affinity for its analyte, uric acid. This biosensor demonstrates high sensitivity and specificity for uric acid, emphasising the effectiveness of green synthesis techniques in advancing biosensor technology. Additionally, it highlights the potential for sustainable innovation by utilizing waste materials.