The main protease (M) is a major protease having an important role in viral replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that caused the pandemic of 2020. Here, active M was obtained as a 34.5 kDa protein by overexpression in BL21 (DE3). The optimal pH and temperature of M were 7.5 and 37 °C, respectively. M displayed a value of 16 μM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified M. The IC values were 137 μg/mL for black garlic extract and 9-197 μM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on M. The structure-activity relationship of these polyphenols revealed that the hydroxyl group in C3', C4', C5' in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on M.