Iris domestica, a perennial herb of the Iridaceae family, is widely recognized for its rich isoflavone content and broad therapeutic properties. To elucidate the biosynthetic pathway of these medicinally significant compounds, we constructed a haplotype-resolved genome assembly of this species. Transcriptomic and metabolomic analyses revealed tissue-specific accumulation of isoflavone, particularly in rhizomes and roots. Functional characterization identified two candidate isoflavone synthase genes, among which IdIFS was confirmed to promote the biosynthesis of key compounds tectorigenin and irisflorentin. The high-quality genome assembly presented here provides a foundational resource for further research into the evolution, secondary metabolite, and environmental adaptation of I. domestica.