In this paper, a novel electrochemical sensor of tetracycline (TC) based on grapheme (GR) /L-cysteine (L-Cys) composite film modified electrode was prepared by using the method of electrode position of the GR and L-cysteine on the glassy carbon electrode (GCE). Meanwhile, the electrochemical behavior of TC was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Compared with GCE, L-Cys/GCE and L-Cys/GR/GCE, the oxidation current on GR/L-Cys/GCE was much higher and the oxidation potential shifted negatively, indicating that the GR/L-Cys/GCE had obviously catalytic activity and electroconductivity on the oxidation of TC. The preparation conditions and experimental parameters were optimized. Under the optimized conditions, the oxidation peak current exhibited a good linear relationship in the concentration range from 8.0 to 140 mu M (R-1 = 0.9893) and the detection limit was 0.12 mu M. The prepared electrode was successfully applied to the determination of TC with the recovery from 89.60% to 105.20%, showing a practical prospect in real samples. (C) 2017 The Electrochemical Society. All rights reserved.