Polyploidy is recognized as a prominent force shaping plant evolution. The autotetraploid I. indigotica with better yield, enhanced resistance and higher lignans accumulation had been obtained in our previous study. However, the causes of novel variation in autotetraploid I. indigotica are not well understood. In the present study, IiWRKY34, which expresses significantly higher in tetraploid I. indigotica than in diploid progenitor, was screened as a major contributor to the control of quantitative traits. Over-expression and RNAi analysis in transgenic I. indigotica hairy roots demonstrated that IiWRKY34 significantly improves root development, salt and drought tolerance, as well as the contents of lignans such as lariciresinol, which represents an important efficacious compound for the antiviral effect of I. indigotica. Integrated analysis of transcriptome and metabolome profiling demonstrated the far-reaching consequences of lignan pathway perturbations on carbon metabolism, phenylpropanoid biosynthesis, and hormone signal transduction, etc. Moreover, IiWRKY34 interacts with Ii4CL3, a key catalytic enzyme involved in lignan pathway of I. indigotica. Meanwhile, IiWRKY34 interacts with IiNAC31, which is predicted as a regulatory factor of stress tolerance. Our study discloses a valuable mechanism for the significantly qualitative difference between tetraploid and diploid I. indigotica by IiWRKY34, and manipulation of this gene should facilitate improvements in yield, stress tolerance and lignan accumulation in I. indigotica and other crops.