MYB transcription factors are widely involved in the regulation of plant growth and development, and they also play important roles in secondary metabolism. In our previous study, the transcription factor TrMYB308 related to flavonoid synthesis was identified by RNA-Seq in V-type purple white clover. Based on these results, the coding sequence (CDS) of TrMYB308 gene was cloned with a length of 921bp, encoding for 306 amino acid residues. Subcellular localization analysis indicated that TrMYB308 was localized to the nucleus. Multiple sequence alignment and phylogenetic analysis indicated that TrMYB308 protein belongs to a typical R2R3-MYB transcription factor subfamily and shows sequence close similarity to TaMYB308 from Trifolium pretense and MtMYB308 from Medicago sativa. Expression analyses revealed that the transcripts of TrMYB3 were detected in all tissues and inducible under JA treatment. Heterologous expression of TrMYB308 in tartary buckwheat hairy root resulted in significant transcriptional elevation of flavonoid metabolic pathway key enzyme genes (FtF3H, FtFLS). Consistently, the content of total flavonoids in transgenic roots was significantly increased if compared to that of the non-transgenic roots. Hence, we speculated that TrMYB308 is involved in the regulation of secondary metabolic biosynthesis of flavonoids. These results provided a theoretical foundation for the genetic improvement of flavonoid content in buckwheat.