Sophora flavescens Aiton is a plant in the Leguminosae family. As a traditional Chinese medicine, it is used to treat eczema, bloody stool, skin pruritus, and so on. By studying non-alkaloid components in the roots of S. flavescens, we obtained a total of 49 compounds (1-49), including three undescribed flavonoids (13, 15 and 18), five undescribed isopentenyl flavonoids (32, 34, 38, 39 and 48), two known coumarins (1-2), three phenolic acids (3-5), one known isopentenyl flavonoids (19-31, 33, 35-37, 40-47 and 49). On the basis of chemical evidences and spectral data analysis (UV, ECD, Optical rotation data, 1D/2D-NMR and HR-ESI-MS), the structures of undescribed compounds were elucidated. The inhibitory effect of compounds 1-49 on LPS induced NO production in RAW 264.7 cells was detected. Compounds 11, 19, 21-24, and 28-30 showed significant inhibitory effects, and the IC50 values of compounds 11 and 22 even reached 4.58 ± 0.66 and 4.53 ± 0.66 μM. This study suggests that flavonoids may be the main component that exerts anti-inflammatory effects in the non-alkaloid extraction layer of the extract from the roots of S. flavescens.