The synthesis of reduced graphene oxide (rGO) is a challenging task for industrial applications due to the absence of a simple and cost-effective synthesis method. This study proposes an environmentally friendly approach to synthesizing rGO using cow urine as a reducing agent, which eliminates the use of hazardous chemicals. The efficacy of this method was confirmed through various analyses, including UV Vis, Fourier transform infrared, and X-ray diffraction, which demonstrated the successful removal of oxygen-containing functions in GO via reduction with cow urine. The synthesized rGO-10 was utilized to fabricate electrochemical nonenzymatic glucose sensors, which exhibited a rapid response time and excellent amperometry response. The sensors used in this study demonstrated a sensitivity of 19.17 μAcm−2mM−1 when optimized for a glucose concentration of 2 mM in human serum. The sensors demonstrated a wide response range, covering a range of 0.2 to 10 mM, and a low detection limit of 1.9019 µM. Additionally, the sensors displayed desirable attributes about repeatability, reproducibility, and stability.