Urea electro-oxidation reaction in alkaline medium is systematically analyzed by electrochemical impedance spectroscopy (EIS). The effects of polarization potential and KOH concentration on the impedance appearance are investigated. In the presence of urea, it is found that Nyquist plots exhibit two depressed semicircles, with one at higher frequencies stably locating in the first quadrant while the other's location at lower frequencies varying between the first and second quadrant as the polarization potential changes. Results show both indirect and direct pathways proceed in urea electro-oxidation reaction. A mathematical model indicates the reverse loop in the Nyquist plots is attributed to CO2 poisoning on the catalyst, which is also validated by the followed chronoamperometric method. Moreover, the rate determining steps of urea electro-oxidation reaction is dependent on KOH concentration. The EIS technique gives a new sight to interpret the poor stability of urea electro-oxidation on nickel catalyst, and thus helps to explore a CO2-insensitive catalyst. (C) 2016 Elsevier Ltd. All rights reserved.