Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a central role in the inactivation of neurotransmitters sharing a catecholic motif by transferring a methyl group from AdoMet. Methylation of the meta-hydroxyl is much more common than that of the para-hydroxyl in many COMT substrates, such as dopamine and norepinephrine. Our experimental data showed that quercetin preferred meta-methylation but luteolin favored a para-methylation. To elucidate the mechanism for different preferences of methylations of quercetin and luteolin, we performed a theoretical investigation on the different regioseletivities of COMT-catalyzed methylations for quercetin and luteolin by a combined approach of MD simulations, ab initio calculations, and QM/MM computations. The ab initio calculation results showed that both quercetin and luteolin have more negative charge distributions on the meta-O atom than the para-O atom, which indicated that meta-O preferred SN2 reaction for their methylation. Our QM/MM computations also confirmed that these two flavonoids have lower reaction energetic barriers for COMT-catalyzed meta-O-methylation than para-O-methylation. On the other hand, our binding free energy computation results indicated that quercetin has a more stable binding mode for meta-O-methylation than para-O-methylation but luteolin has a more stable binding mode for para-O-methylation than meta-O-methylation. We gave a comprehensive explanation considering both thermodynamics and reaction kinetics aspects and discussed the protein-inhibitor interactions as well as the O-methylation mechanism in our present work.