Uniform sputtered nanoporous zinc oxide (Nano-ZnO) thin film on the conductive fluorinated-tin oxide (FTO) layer was applied to immobilize urease enzyme (Urs) for urea detection. Highly uniform nanoporous ZnO thin film were obtained by reactive radio frequency (RF) magnetron sputtering system at the optimized instrumental deposition conditions. Characterization of the surface morphology and roughness of ZnO thin film by field emission-scanning electron microscopy (FE-SEM) exhibits cavities of nanoporous film as an effective biosensing area for enzyme immobilization. Step by step monitoring of FTO/Nano-ZnO/Urs biosensor fabrication were performed using electrochemical methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Fabricated FTO/Nano-ZnO/Urs biosensor was used for urea determination using EIS experiments. The impedimetric results show high sensitivity for urea detection within 0.83-23.24 mM and limit of detection as 0.40 mM. (C) 2014 Elsevier Ltd. All rights reserved.