An ultrasensitive electrochemical sensing platform based on three-dimensional (3D) graphene-like carbon architecture (3D-HPG) and polythionine (PTH) modified glassy carbon electrode (GCE) was developed for the determination of metronidazole. The 3D-HPG/PTH interface possesses 3D porous structure, large electro-active surface area and excellent electrical conductivity. Electrochemical behavior of metronidazole at the 3D-HPG/PTH/GCE was investigated by cyclic voltammetry (CV), and it has been found that the 3D-HPG/PTH nanocomposite exhibited synergistic electro-catalytic property for the reduction of metronidazole. Under the optimized conditions, metronidazole was determined by CV and differential pulse voltammetry (DPV). DPV was found to be more effective for detecting low concentrations of metronidazole. Two linear ranges of 0.05-70 mu M and 70-500 mu M were obtained, with a detection limit of 1 nM (S/N = 3). The 3D-HPG/PTH/GCE exhibits high sensitivity, good selectivity, satisfactory reusability and reproducibility. It was applied for the determination of metronidazole in real water samples, with satisfactory recoveries ranging from 92.8% to 103%, and was successfully applied for the analysis of pharmaceutical dosage form. (C) 2018 The Electrochemical Society.