Background Astragalus membranaceus(Fisch.) Bge. var.mongholicus(Bge.) Hsiao is a traditional medicinal herb of Leguminosae since it contains bioactive compounds such as flavonoids, which have significant pharmacological effects on immunity and antioxidant. However, the scanty genomic and transcriptome resources ofAstragalus membranaceushave hindered further exploration of its biosynthesis and accumulation mechanism. Objective This project aim to further improve our understanding of the relationship between transcriptional behavior and flavonoids content ofA. mongholicus. Methods The accumulation of flavonoids and related gene expression in five different developmental stages (A: vegetative, B: florescence, C: fruiting, D: fruit ripening and E: defoliating stages) ofA. mongholicusroot were studied by combining UV spectrophotometry and transcriptomic techniques. Thede novoassembly, annotation and functional evaluation of the contigs were performed with bioinformatics tools. Results After screening and assembling the raw data, there were a total of 158,123 unigenes with an average length of 644.89 bp were finally obtained, which has 8362 unigenes could be jointly annotated by NR, SwissProt, eggNOG, GO, KEGG and Pfam databases. KEGG enrichment analysis was performed on differentially expressed genes(DEGs)in the four groups (A vs. B, B vs. C, C vs. D, D vs. E). The results showed that many DEGs in each group were significantly enriched to flavonoids biosynthesis related pathways. Among them, a number of 86 were involved in the biosynthesis of isoflavonoid (12), flavonoid (5) and phenylpropanoid (69). Further analysis of these DEGs revealed that the expression levels of key genes such asPAL,4CL,CCR,COMT,DFR, etc.were all down-regulated at the fruiting stage, and then raised at the fruit ripening stage. This expression pattern was similar to the accumulation trend of total flavonoids content. Conclusions In summary, this comprehensive transcriptome dataset allowed the identification of genes associated with flavonoids metabolic pathways. The results laid a foundation for the biosynthesis and regulation of flavonoids. It also provided a scientific basis for the most suitable harvest time and resource utilization ofA. mongholicus.