Descurainia sophia Willd. belongs to the genus Descurainia of Cruciferae. Its dried, mature seeds are known as “Tinglizi” and are widely used in traditional Chinese medicine. The seeds of D. sophia are rich in unsaturated fatty acids, glucosinolates, flavonoids, and a variety of compounds with biological activity. In order to obtain detailed information on the genome of D. sophia and to explore the molecular basis of glucosinolate biosynthesis in D. sophia. We assembled a high-quality telomere-to-telomere (T2T) reference genome of the D. sophia genome (2n=4x=28) using PacBio and ONT long-reads, in combination with Illumina short-read sequencing and Hi-C technology. The assembled genome was 266.23 Mb in size, with 36.12% GC content and 23.28% repeat content. There are 14 pseudo-chromosomes in the Hi-C-assembled genome sequence. A total of 57,590 genes and 87 pseudogenes were predicted, 98.94% of which can be annotated to Nr, GO, KEGG, TrEMBL, Swissport, Pfam, and InterPro databases. Comparative genomic analyses indicated that the expanded gene families were significantly involved in sesquiterpenoid and triterpenoid biosynthesis, homologous recombination, and inositol phosphate metabolism. Moreover, we identified 55 genes involved in glucosinolate biosynthesis in D. sophia. UGT74B1 and SOT16 catalyze the final two steps of glucosinolate biosynthesis; we cloned these genes and verified their functions using in vitro catalysis and transgenic A. thaliana. Over-expression of the DsUGT74B1 gene can significantly promote the growth of A. thaliana by regulating IAA and BR signal pathways, and the content of glucosinolates and flavonoids in the transgenic plants was remarkably higher than that in the wild-type A. thaliana. The high-resolution reference genome generated by this study will provide accurate sequence information for glucosinolate biosynthesis research of D. sophia.