N2 incorporated diamond nanowire (N-DNW) film electrochemical biosensor has utilized for the quantitative determination of urea in aqueous solution and urine sample. N-DNW electrode is wet-chemically cleaned (oxidation) by boiling in a mixture of H2SO4 and HNO3 (3:1) at 200 °C for 2 h to remove graphite. Urease (Urs) and glutamate dehydrogenase (GLDH) are covalently attached to the oxidized N-DNW electrode by activating the COOH group of N-DNW using ethyl(dimethylaminopropyl)carbodiimide as the coupling agent and N-hydroxysuccinimide as activator. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy data reveal that carboxylic and hydroxyl functionalized nature of N-DNW electrodes Urs–GLDH immobilized N-DNW (Urs–GLDH/N-DNW) has been successfully utilized in urea biosensor which exhibits good performance in sensitivity (6.18 μA/mg dL/cm2), stability (~1 month), reproducibility, lower detection limit (3.87 mg/dL) and fast response time (>10 s). Urs–GLDH/N-DNW also exhibits electrochemical response when tested for different concentration of human urine in buffer solution (from 1:9 to 4:6). In addition, Urs–GLDH/N-DNW bioelectrode retains 80% of its initial enzyme activity for <1 month, when stored at 4–6 °C in a refrigerator.