The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone was studied in the presence of laccase from Pyricularia oryzae. The findings show that laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) catalyzes the oxidative coupling reaction between MBTH and phenols producing red colored ate-dye compounds. On the basis of kinetic parameters and optimum pH values, the mechanisms of the oxidative coupling reactions with the different phenols are discussed. The results suggest that the reaction is initialized by the enzymatic 3-methyl-2-benzothiazolinone hydrazone activation, which undergoes electrophilic substitution with m-methoxyphenol in solution, enzymatic activated guaiacol, and chelated p-methoxyphenol at the catalytic site of the laccase. The current study also demonstrates the possibility of using the ate-dye formed in the presence of guaiacol for measuring laccase activity following a similar assaying method as that used for measuring peroxidase activity. This type of assay permits the determination of laccase at micromolar levels by fixed time method.