Fabrication of new advanced nonenzymatic electrochemical nano-sensors of glucose has recently attracted intensive attention. In this work, we designed a novel ultrasensitive nonenzymatic amperometric sensor for detection of glucose by incorporating two pairs of effective electron mediators, Ni(II)/Ni(III) and Au/Au(I), into a nanoporous structure, namely a nanoporous gold film (NPGF) coated with a thin layer of nickel hydroxide about 4 nm in thickness. The NPGF with high roughness was quickly prepared by anodic potential step, and the thin surface coating of Ni(OH)(2) was easily obtained by electrooxidizing the electrodeposited Ni coverlayer. The incorporation of thin Ni(OH)(2) coating into/on the NPGF led to mutual stabilization without changing the nanoporous structure. The Ni(OH)(2)/NPGF electrode fabricated totally by facile electrochemical methods at room temperature showed high electrocatalytic activity for the oxidation of glucose within a wide potential range (-0.5 similar to 0.2 V) due to co-mediating of the two pairs of electron mediators including their coupling Ni(III) + Au = Ni(II) + Au(I) The electrode also demonstrated excellent performance in sensing glucose concentration with a wide linear range(2 mu M similar to 7 mM), ultrasensitivity (3529 mu A mM(-1) cm(-2)), low detection limit (0.73 mu M), good repeatability, and long-term stability (3 weeks), which was successfully applied to detect glucose in a human blood serum sample by standard addition method with satisfactory recovery. This work is expected to open a new avenue to fabricate nonenzymatic electrochemical sensors of glucose involving co-mediating. (C) 2014 Elsevier Ltd. All rights reserved.