Abstract: Acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a global pandemic since 2019, and posed a serious threat to global health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components originated from TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which results in the characterization of 12 new flavonoids, including seven prenylated flavanones ( 1 − 7 ), one prenylated flavonol ( 8 ), two prenylated chalcones ( 9 − 10 ), one isoflavanone ( 11 ), and one isoflavan dimer ( 12 ), together with 43 known compounds ( 13 − 55 ). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC₅₀ values of 34.89 and 19.88 μmol·L⁻¹, repectively while compounds 9 , 43 and 47 exhibited inhibitory effects against PLpro with IC₅₀ values of 32.67, 79.38, and 16.74 μmol·L⁻¹, respectively.