Sustainable and eco-friendly biosensing technologies are emerging as a potential approach for healthcare improvement while simultaneously reducing environmental impact. In this study, a novel uric acid biosensor has been developed using green-synthesized molybdenum oxide (MoO3) thin films, which demonstrates a creative way to repurpose waste for advanced technological applications. The structural and morphological studies confirmed the uniformity and high quality of the MoO3 thin films, which played a key role in enhancing sensor performance. The fabricated biosensor demonstrated high sensitivity [244.76 µA/(mM cm2)], a fast response time of 5 s, and excellent selectivity against common interferents which are typically found in blood plasma. Additionally, the low Michaelis–Menten constant (Kₘ = 0.07311 mM) indicates that the enzyme maintained a strong affinity for uric acid, ensuring reliable detection even at low concentrations. In addition to its strong analytical performance, this work highlights the potential of green synthesis in the development of biosensors. By integrating eco-conscious material synthesis with high-performance sensing capabilities, this work lays the foundation for more sustainable, cost-effective, and efficient biosensors for healthcare applications. This approach not only enhances biosensing technology but also aligns with global efforts to minimize waste and promote greener alternatives in scientific innovation.