In the present study, a new biosensor based on laccase from Paraconiothyrium variabile was developed for catechol. The purified enzyme entrapped into the Fe3O4/polyaniline/chitosan (Fe3O4/polyaniline (PANI)/chitosan (CS)) biocomposite matrix film without the aid of other cross-linking reagents by a one-step electrodeposition on the surface of carbon paste electrode (CPE). The formed layer of biocomposite was characterized with scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The biosensor was optimized with respect to biocomposite composition, enzyme loading, and solution pH by amperometry method. The biosensor exhibited noticeable eletrocatalytic ability toward catechol with a linear concentration range from 0.5 to 80 mu M and a detection limit of 0.4 mu M. The biosensor showed optimum response within 8 s, at pH 5, and 40 A degrees C. The apparent Michaelis-Menten (K (M) (app)) was found to be 1.092 mu M. The fabricated biosensor could be applied for determination of catechol in tea leaf samples.