Tartary buckwheat (Fagopyrum tataricum) is a dual-purpose medicinal and food crop grown for its high contents of functional compounds and abundant nutrients. Although studies have shown the differences of total flavonoid content in Tartary buckwheat at different ploidy levels, the composition of flavonoid and its regulatory mechanisms are largely unknown. In this study, the leaf metabolome and transcriptome of diploid and tetraploid accessions of Tartary buckwheat were analyzed to gain insight into the impact of polyploidization on comparative secondary metabolite composition and molecular regulatory mechanism. Based on a widely targeted metabolomics analysis, a total of 792 metabolites were identified, including 127 flavonoids. The accumulation of 127 metabolites and expression of 3871 genes differed significantly between diploid and tetraploid Tartary buckwheat. Integrated metabolomics and transcriptome analysis revealed that chromosome doubling up-regulated the expression of upstream genes in the flavonoid biosynthesis pathway to promote the accumulation of flavonoids. The present results contribute to elucidation of the molecular mechanism of phenotypic variation associated with polyploidy in Tartary buckwheat. The findings provide a reference for further studies on phenotypic traits in polyploid Tartary buckwheat, the cloning of crucial regulatory genes, and utilization of genetic engineering technologies in the breeding of Tartary buckwheat.