Ginkgo biloba is the oldest relict plant on Earth and an economic plant resource derived from China. Flavonoids extracted from G. biloba are beneficial to the prevention and treatment of cardiovascular and cerebrovascular diseases. Basic leucine zipper (bZIP) transcription factors (TFs) have been recognized to play important roles in plant secondary metabolism. In this study, GbbZIP08 was isolated and characterized. It encodes a protein containing 154 amino acids, which belongs to hypocotyl 5 in group H of the bZIP family. Tobacco transient expression assay indicated that GbbZIP08 was localized in the plant nucleus. GbbZIP08 overexpression showed that the contents of total flavonoids, kaempferol, and anthocyanin in transgenic tobacco were significantly higher than those in the wild type. Transcriptome sequencing analysis revealed significant upregulation of structural genes in the flavonoid biosynthesis pathway. In addition, phytohormone signal transduction pathways, such as the abscisic acid, salicylic acid, auxin, and jasmonic acid pathways, were enriched with a large number of differentially expressed genes. TFs such as MYB, AP2, WRKY, NAC, bZIP, and bHLH, were also differentially expressed. The above results indicated that GbbZIP08 overexpression promoted flavonoid accumulation and increased the transcription levels of flavonoid-synthesis-related genes in plants.