Impatiens noli-tangere accumulates abundant α-linolenic acid (ALA) and its metabolic volatiles, which hold significant potential for applications in healthcare and agriculture. However, the genetic basis underlying their biosynthesis has not been systematically investigated. Here, we present a high-quality genome assembly for I. noli-tangere (614.46 Mb). Despite a high repetitive sequence content (70.46%), it avoided excessive expansion due to the efficient elimination of long terminal repeat retrotransposons. Phylogenomic analyses revealed that I. noli-tangere experienced two whole-genome duplication (WGD) events, with WGD-derived genes predominating in oil biosynthesis. Notably, IntFAD3, a WGD-duplicated fatty acid desaturase, was identified as a key seed-specific gene for ALA biosynthesis. Its regulation by the transcription factor IntbZIP38 was functionally validated through yeast one-hybrid, luciferase, β-glucuronidase, and transgenic functional assays. Furthermore, (E)-2-hexenal, the predominant ALA-derived volatile in leaves, exhibited potent antifungal activity against Botrytis cinerea (minimum inhibitory concentration: 0.188 ml/l), with its biosynthesis linked to Int13-HPL. These findings provide genomic and functional insights into ALA biosynthesis and metabolic volatiles in I. noli-tangere, supporting its potential in sustainable agriculture and bioactive compound development.

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