Herein, a novel strategy for electrochemical detection of NADH was developed. The electrode was modified by 3-aminophenylboronic acid functionalized multiple-walled carbon nanotubes (MWCNTs), and then phenazine methosulfate (PMS) acting as shuttle mediators was stably absorbed on the MWCNTs through pi-pi stacking. The enrichment of NADH on the surface of the modified electrode was achieved through boronate affinity between boronic acid and NADH containing cis-diol group. PMS absorbed on the MWCNTs promoted the electron transfer between NADH and the electrode surface at -0.10 V (vs. Ag/AgCl) with a good sensitivity of 161 mu A mmol L-1 cm(-2) and a detection limit of 0.16 mu mol L-1 (S/N = 3) in amperometric response. The proposed electrochemical sensors displayed a wide linear response from 0.5 mu mol L-1 to 1.0 mmol L-1 NADH with a fast response time of less than 3 s, and allowed the selective detection in the presence of ascorbic acid, uric acid, dopamine and glucose. Furthermore, the fabricated sensor was applied to the determination of NADH in human serum. The present method was promising for development of dehydrogenase-based biosensors. (C) 2017 Elsevier B.V. All rights reserved.