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<i>Ginkgo biloba</i> is a highly valuable medicinal plant known for its rich secondary metabolites, including flavonoids. Zinc oxide nanoparticles (ZnO-NPs) can be used as nanofertilizers and nano-growth regulators to promote plant growth and development. However, little is known about the effects of ZnO-NPs on flavonoids in <i>G. biloba</i>. In this study, <i>G. biloba</i> was treated with different concentrations of ZnO-NPs (25, 50, 100 mg/L), and it was found that 25 mg/L of ZnO-NPs enhanced <i>G. biloba</i> fresh weight, dry weight, zinc content, and flavonoids, while 50 and 100 mg/L had an inhibitory effect on plant growth. Furthermore, quantitative reverse transcription (qRT)-PCR revealed that the increased total flavonoids and flavonols were mainly due to the promotion of the expression of flavonol structural genes such as <i>GbF3H</i>, <i>GbF3′H,</i> and <i>GbFLS</i>. Additionally, when the <i>GbF3H</i> gene was overexpressed in tobacco and <i>G. biloba</i> calli, an increase in total flavonoid content was observed. These findings indicate that 25 mg/L of ZnO-NPs play a crucial role in <i>G. biloba</i> growth and the accumulation of flavonoids, which can potentially promote the yield and quality of <i>G. biloba</i> in production.