Abstract Methotrexate (MTX), a common pharmaceutical drug in cancer therapy and treatment of rheumatic diseases, is known to cause severe adverse side effects at high dose. As the side effect may be life threatening, there is an urgent need for a continuous, bed-side monitoring of the nominal MTX serum level in a patient while the chemical is being administered. This article describes a detection of MTX using a flow system that consists two modified gold electrodes. Interaction of MTX with the antibodies fixed on the electrode surface is detected by electrochemical impedance spectroscopy and evaluated using singular value decomposition (SVD). The key finding of this work is that the change in the electrode capacitance is found to be quantitative with respect to the concentration of MTX. Moreover a calibration curve constructed using the principal component regression method has a linear range of six orders of magnitude and a detection limit of 1.65 × 10−10 M.