Sea buckthorn is a plant with similarities to both food and medicine, possessing abundant flavonoids. However, the metabolic mechanism of flavonoid synthesis in sea buckthorn is unclear. Here, we performed transcriptomic and metabolomic analyses to explore the metabolic characteristics of flavonoids in different sea buckthorn tissues and their associated gene regulatory networks. A total of 441 metabolites were identified from sea buckthorn by metabolomics (UPLC-MS/MS), which includes 104 flavonoids with significant differences in the relative contents among tissues. Transcriptomic analysis identified 75 genes involved in the flavonoid biosynthesis pathway. The weighted gene correlation network analysis revealed that the MEdarkgreen module showed a significant correlation with flavonoid compounds and comprised ten genes related to flavonoid synthesis. Overexpression of HrMYB114 resulted in a significant increase in flavonoid content and expression of structural genes in the flavonoid synthesis pathway in transgenic hairy roots. This study provided a comprehensive analysis of metabolic and transcriptomic profiles, which helped to investigate the molecular mechanism of flavonoid biosynthesis.