The preparation, characterization and application of histidine (His) modified montmorillonite (His-Mt) in electrochemical biosensor fabrication were reported. His was used as a modifier to enhance immobilization capability of Mt for the biomolecules. Mt modifications were followed by X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetry analysis. After a successful immobilization of Laccase enzyme on the His-Mt matrix, covered on a glassy carbon electrode, application in catechol biosensing was investigated in detail. Optimization, analytical characteristics, repeatability, and operational stability studies were evaluated using both batch and flow injections analysis configurations. (C) 2013 Elsevier B.V. All rights reserved.