A metal-ligand dual-site single-atom nanozyme (Ni-DAB) was developed to mimic urate oxidase (UOX) with high specificity for uric acid (UA) oxidation. The Ni-DAB nanozyme features a Ni metal center and a beta C atom in the ligand as specific binding sites for UA and O₂, respectively. This dual-site structure enables high catalytic specificity, as confirmed by experimental and theoretical studies. The nanozyme exhibits UOX-like activity, efficiently oxidizing UA with high specificity and stability under various pH and temperature conditions. It was further demonstrated that Ni-DAB can be used in a biofuel cell powered by human urine, successfully driving a temperature and humidity sensor. The study highlights the potential of mimicking natural enzyme mechanisms to enhance the selectivity of non-protein artificial enzymes. The Ni-DAB nanozyme shows superior performance compared to other catalysts, with a specific activity of 1.51 U mg⁻¹. The results suggest that the dual-site design is crucial for achieving high catalytic specificity, and the nanozyme has promising applications in biofuel cells and other enzymatic mimics.A metal-ligand dual-site single-atom nanozyme (Ni-DAB) was developed to mimic urate oxidase (UOX) with high specificity for uric acid (UA) oxidation. The Ni-DAB nanozyme features a Ni metal center and a beta C atom in the ligand as specific binding sites for UA and O₂, respectively. This dual-site structure enables high catalytic specificity, as confirmed by experimental and theoretical studies. The nanozyme exhibits UOX-like activity, efficiently oxidizing UA with high specificity and stability under various pH and temperature conditions. It was further demonstrated that Ni-DAB can be used in a biofuel cell powered by human urine, successfully driving a temperature and humidity sensor. The study highlights the potential of mimicking natural enzyme mechanisms to enhance the selectivity of non-protein artificial enzymes. The Ni-DAB nanozyme shows superior performance compared to other catalysts, with a specific activity of 1.51 U mg⁻¹. The results suggest that the dual-site design is crucial for achieving high catalytic specificity, and the nanozyme has promising applications in biofuel cells and other enzymatic mimics.