Vegetable oils rich in polyunsaturated fatty acids (PUFAs), such as Paeonia ostii seed oil (>70% PUFAs), have significant nutritional and health benefits. Flavonoids, while beneficial to human health, also influence oil quality. However, the mechanisms governing flavonoid accumulation in seeds and their relationship with PUFA biosynthesis remain unclear. In this study, we analyzed the total flavonoid content across 124 Paeonia cultivars and selected five superior varieties. Among them, P. ostii ‘Fengdan’ (P. ostii ‘FD’) underwent wide-target metabolomic analysis, revealing 544 differentially accumulated secondary metabolites, including 219 flavonoids—63% of which were glycoside derivatives. Transcriptomic analysis identified key genes involved in flavonoid biosynthesis, focusing on PoUGT84A1, a UDP-glycosyltransferase. In vitro enzymatic assays confirmed that PoUGT84A1 glycosylates quercetin, kaempferol, and luteolin, enhancing their stability. Notably, both overexpression and silencing of PoUGT84A1 altered flavonoid and PUFA accumulation. Virus-induced gene silencing (VIGS) of PoUGT84A1 in P. ostii ‘FD’ seeds reduced its expression to 19.06% of TRV control levels, slightly increased quercetin-3-O-glucoside (Qu3G) content (12.26%) and significantly decreased fatty acid (FA) levels—by 59.57%, 56.66%, and 57.44% for oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA), respectively—compared to TRV controls. These findings advance our understanding of plant lipid metabolism and offer a promising genetic strategy for improving oil crops. This research provides valuable insights for developing high-quality vegetable oils enriched in PUFAs and flavonoids, with implications for human health, nutrition, and the sustainable oil crop industry.