Aconitum carmichaelii Debx. is a herbal species that contains many precious bioactive substances, which are alkaloids, flavonoids, steroids, and glycosides. Flavonoids, which are major secondary compounds, play an important role in maintaining redox balance in the cells of the plant body. Many flavonoids have antibacterial, antioxidant, and anticancer properties. However, studies have mainly focused on aconitine, which is a highly toxic group A poison belonging to the alkaloid group, but with little mention of flavonoids. The flavonoids in A. carmichaelii are a group of substances with high content, concentrated in leaves and flowers, including quercetin and kaempferol. F3′5′H (Flavonoid 3′5′-hydroxylase) has been identified as the key enzyme involved in the final steps of flavonoid biosynthesis in plants in general and in A. carmichaelii specifically. This study offers the first report, and demonstrates that the overexpression of the F3′5′H gene from a herbal plant, A. carmichaelii, increases flavonoid content in genetically modified tobacco plants. The A. carmichaelii gene was transformed into tobacco leaf tissue to create transgenic tobacco plants. The AcF3′5′H gene was incorporated into the tobacco genome and was expressed in four transgenic tobacco lines (T01, T03, T05, and T014). The F3′5′H content increased from 20.33% to 32.00% compared with that in non-transformed plants (P < 0.001). Therefore, the flavonoid content of four transgenic tobacco lines increased compared to the WT, from 69.23% to 122.23% (P < 0.001). The results of the successful expression of the AcF3′5′H gene in model tobacco plants are the basis for using the AcF3′5′H gene for improving flavonoid content in other medicinal plants. Thus, the AcF3′5′H gene considered in this work could be a candidate for gene technology to enhance flavonoid accumulation in plants.