This study reports the development of a metal-ligand dual-site single-atom nanozyme (Ni-DAB) that mimics urate oxidase (UOX) for selective oxidation of uric acid (UA). Ni-DAB, composed of Ni metal centers and 3,3'-diaminobenzidine (DAB) ligands, exhibits high catalytic specificity for UA oxidation. The Ni-DAB structure was characterized using various techniques, including X-ray absorption spectroscopy, X-ray photoelectron spectroscopy, and isotope labeling. The catalytic mechanism of Ni-DAB was elucidated through theoretical calculations, revealing that the Ni center binds UA, while the beta C atoms in the DAB ligand bind O2. This dual-site approach enhances the selectivity of non-protein artificial enzymes, similar to natural enzymes. A biofuel cell using human urine as a source of UA was constructed, demonstrating the practical application of Ni-DAB in energy harvesting. The study provides a novel strategy for improving the selectivity of artificial enzymes by mimicking the catalytic mechanisms of natural enzymes.This study reports the development of a metal-ligand dual-site single-atom nanozyme (Ni-DAB) that mimics urate oxidase (UOX) for selective oxidation of uric acid (UA). Ni-DAB, composed of Ni metal centers and 3,3'-diaminobenzidine (DAB) ligands, exhibits high catalytic specificity for UA oxidation. The Ni-DAB structure was characterized using various techniques, including X-ray absorption spectroscopy, X-ray photoelectron spectroscopy, and isotope labeling. The catalytic mechanism of Ni-DAB was elucidated through theoretical calculations, revealing that the Ni center binds UA, while the beta C atoms in the DAB ligand bind O2. This dual-site approach enhances the selectivity of non-protein artificial enzymes, similar to natural enzymes. A biofuel cell using human urine as a source of UA was constructed, demonstrating the practical application of Ni-DAB in energy harvesting. The study provides a novel strategy for improving the selectivity of artificial enzymes by mimicking the catalytic mechanisms of natural enzymes.