Atractylodes chinensis (DC.) Koidz is a pharmacologically significant medicinal plant that produces various bioactive metabolites, including β-eudesmol, which largely determines its medicinal quality and clinical efficacy. Treatment of A. chinensis hairy roots with 5-methoxyindole (5-MI), a chemical homolog of melatonin, revealed that 0.5 mmol·L⁻¹ 5-MI significantly enhanced β-eudesmol production. To investigate the underlying mechanism, we performed integrated transcriptomic and metabolomic analyses. Comprehensive targeted metabolomics identified ten upregulated terpenoids among the differentially expressed metabolites following 5-MI treatment in A. chinensis hairy roots, including β-eudesmol. The 5-MI treatment significantly influenced the expression levels of genes associated with metabolic regulation and secondary metabolite synthesis in A. chinensis hairy roots, particularly promoting the upregulation of genes involved in terpenoid biosynthesis. Notably, a putative sesquiterpene synthase gene, AcTPS1, was significantly upregulated after 5-MI treatment and was a strong candidate gene correlated with β-eudesmol. AcTPS1 belongs to the TPS-a subfamily and exhibits tissue-specific expression, with high transcript levels in root tissues, especially in one-year-old roots of A. chinensis. This study demonstrates that 5-MI serves as an effective elicitor, providing valuable insights for the production of medicinally active compounds and enhancing our understanding of the molecular mechanism by which 5-MI regulates plant secondary metabolism.