The flavonoids in Ginkgo biloba L. (ginkgo) have important medicinal uses due to their antioxidant, antitumor, and blood circulation-promoting effects. However, the genetic mechanisms underlying flavonoid biosynthesis in ginkgo remain elusive. Flavonoid 3′, 5′-hydroxylase (F3′5′H) is an important enzyme in flavonoid synthesis. We detected a novel differentially expressed GbF3′5′H1 gene homologous to the F3′5′H enzyme involved in the flavonoid synthesis pathway through transcriptome sequencing. In this study, we characterized this gene, performed an expression analysis, and heterologously overexpressed GbF3′5′H1 in Populus. Our results showed that GbF3′5′H1 is abundant in the leaf and highly expressed during April. We also found four metabolites closely related to flavonoid biosynthesis. Importantly, the contents of 4′,5-dihydroxy-7-glucosyloxyflavanone, epicatechin, and gallocatechin were significantly higher in transgenic plants than in nontransgenic plants. Our findings revealed that the GbF3′5′H1 gene functions in the biosynthesis of flavonoid-related metabolites, suggesting that GbF3′5′H1 represents a prime candidate for future studies (e.g., gene-editing) aiming to optimize ginkgo flavonoid production, especially that of flavan-3-ols.