A highly sensitive electrochemical biosensor for the detection of trace amounts of methotrexate had been designed. Gold nanoparticles were allowed to self-assemble onto a glassy carbon electrode that was prior modified by L-cysteine. The anodic voltammetric behaviors of methotrexate at the electrode were then explored by cyclic voltammetry (CV) and square wave voltammetry (SWV). Compared with bare GCE, the electrode can greatly improve the measuring sensitivity of methotrexate. More, the dependences of the current on pH, nature of buffer, instrumental parameters, accumulation time and potential were investigated to optimize the experimental conditions for the determination of methotrexate. Under the selected conditions, the modified electrode in pH = 2.0 Britton-Robinson (B-R) buffer solutions showed a linear voltammetric response to methotrexate within the concentration range of 4.0 x 10(-8) - x 10(-6) mol L-1, with the detection limit of 1.0 x 10(-8) mol L-1. The method was applied to detect methotrexate in medicinal tablets and spiked human blood serum samples. (C) 2012 Elsevier B.V. All rights reserved.