A highly sensitive electrochemical biosensor for the detection of trace amounts of methotrexate has been designed. Double stranded (ds)DNA molecules are immobilized onto a pretreated glassy carbon electrode (GCE(ox)) surface with Langmuir-Blodgett (LB) technique. The adsorptive voltammetric behaviors of methotrexate on DNA-modified electrode were explored by means of cyclic voltammetry (CV) and square wave voltammetry (SWV). The oxidation mechanism was proposed and discussed in this work. In addition, the optimum experimental conditions for the detection of methotrexate were explored, and the currents measured by SWV presented a good linear property as a function of the concentrations of methotrexate in the range of 2.0 x 10(-8) to 4.0 x 10(-6) mol L-1, with an LOD of 5.0 x 10(-9) mol L-1. The method proposed was applied for the determination of methotrexate in pharmaceutical dosage and diluted human urine with wonderful satisfactory successfully. (C) 2008 Published by Elsevier Ltd