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.