Urea oxidation reaction (UOR) has been known as a viable method for renal/liver disease diagnostic detection. Here, we reported a three-dimensional (3D) nickel oxide nanoparticles dressed carbonized eggshell membrane (3D NiO/c-ESM) as a modified electrode toward urea detection. Several common physical measurements were employed to confirm its structural and morphological information. NiO/c-ESM modified electrode exhibits an outstanding performance for urea determination with a linear range from 0.05 to 2.5 mM, and limit detection of ~20 μM (3σ). This work offered a green approach for introducing 3D nanostructure through employing biowaste ESMs as templates, providing a typical example for producing new value-added nanomaterials with urea detection.