This work describes the electrochemical behavior of Ni-2,3-pyrazine dicarboxylic acid (Ni-PDA) film immobilized on the surface of penicillamine (PNA)/CdS-quantum dots (CdS-QDs) gold nanoparticle (AuNPs) Au electrode and its electrocatalytic activity toward oxidation of urea. The surface structure and composition of the sensor was characterized by scanning electron microscopy (SEM). Oxidation of urea on the surface of modified electrode was investigated with cyclic voltammetry, electrochemical impedance spectroscopy (EIS), hydrodynamic voltammetry and chronoamperometry methods and the results showed that the PNA/CdS-QDs/PDA-Ni film displays excellent electrochemical catalytic activities towards urea oxidation. The modified electrode indicated reproducible behavior and high level of stability during the electrochemical experiments. Also it has short response time, low detection limit, high sensitivity and low operation potential. It can be used as an amperometric sensor for monitoring of urea. The proposed modified electrode was successfully used for determination of urea in real sample such human serum. (C) 2014 Elsevier Ltd. All rights reserved.