Nitrogen doped carbon sphere (NDCS), with an average diameter ranging from 220 to 280, was prepared with glucose and melamine mixture as starting materials and ethylene glycol as the solvent and reducing agent by solvothermal method. The microstructures of the NDCS were characterized by X-ray photoelectron spectroscopy (XPS), XRD pattern and Raman spectra. NDCS exhibited highly electrocatalytic activity towards the oxidation of biomolecules such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) due to its unique structure and properties originating from nitrogen doping. This new finding provides a novel and promising candidate of advanced electrode material for the simultaneous determination of AA, DA and UA in human metabolism.