In this paper, a simple and highly selective electrochemical method for the simultaneous determination of catechol (CC) and hydroquinone (HQ) has been developed with a three-dimensional (3D) N-doped carbon nanotube (NCNT) film electrode. The 3D NCNT film was prepared by the combination of electrospinning and chemical vapor deposition procedure; dense and uniform NCNTs were firmly bonded onto the electrospun carbon nano fiber matrix (NCNT@CNFs). By directly dropping the flexible film onto the electrode surface without additional oxidant treatment, a dihydroxybenzene biosensor can be easily constructed. Differential pulse voltammetry (DPV) results showed that the isomers can be detected selectively at NCNT@CNF modified electrode with peak-to-peak separation about 115 mV. Under the optimized condition, the sensing platform showed wide linear responses from 0.08 to 350 mu M and 0.1-425 mu M with detection limits of 20 nM and 50 nM (S/N = 3) for CC and HQ respectively. The proposed method was successfully applied to the simultaneous determination of CC and HQ in real samples with reliable recovery. The N-doping combining with abundant defective sites and favorable 3D network structure facilitate the electron transfer, which resulted in excellent electrocatalytic performance. The attractive electrochemical performances and facile preparation method made this novel electrode promising for the development of effective dihydroxybenzene sensor. (C) 2015 Published by Elsevier B.V.