Grona styracifoliais a photophilous legume that contains abundant flavonoids with multiple pharmacological activities, which is used to cure urethral and biliary calculus in China for thousands of years. The authentication of the rate-limiting enzymes involved in the flavonoids biosynthesis pathway enabled a better understanding of the molecular aspect of quality formation and modulation of this medicinal herb. In this study, the chemical distribution characteristics and content of flavonoids in different tissues ofGrona styracifoliawere analyzed using ultraperormance liquid chromatography coupled with Q-TOF mass spectrometry and showed that active flavonoids were primarily synthesized and stored in the leaves. Subsequently, RNA sequencing (RNA-seq)-based transcriptome profiling of the different tissues revealed that the flavonoids biosynthesis in the leaves was the most active. Meanwhile, 27 full-length transcripts inferred encoding vital enzymes involved in the flavonoids biosynthesis were preliminarily excavated. Finally, four CHSs, four CHIs, and one FNSII were successfully characterized by heterologous expression, which involved in three rate-limiting steps of the flavonoid biosynthetic pathway. In conclusion, these results laid a foundation for further investigation of the molecular mechanism of the biosynthesis and modulation of active flavonoids inGrona styracifolia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04290-z.