Ginkgo biloba L. leaves are rich in secondary metabolites with important medicinal values; to increase their contents, foliar spraying of micronutrients is a potential strategy. Zinc, a multifunctional element, has a significant impact on the content of secondary metabolites in other plants, but relevant research into ginkgo is still lacking. In our study, different spraying time and concentration strategies were used to investigate the effects of zinc sulfate (ZnSO4) on physiological indicators and secondary metabolites of 2-year-old ginkgo. The results demonstrated that ZnSO4 could increase the contents of hydrogen peroxide, abscisic acid, and free amino acids in ginkgo leaves. It also enhances the antioxidant enzyme activity of ginkgo leaves, decreases the content of plant auxin, and ultimately facilitates the accumulation of ginkgo terpene lactones (TTL). Spraying ZnSO4 in June resulted in a more significant increase in the contents of TTL and flavonoids compared to spraying in August. After spraying 12 mmol/L ZnSO4 in June, the contents of TTL and flavonoids in ginkgo leaves were significantly elevated by 35.95% and 24.30%, respectively, compared to those in the CK (p < 0.05). The contents of ginkgolide A, B, and C were notably increased by 45.93%, 46.56%, and 74.29%, respectively, compared to those in the CK (p < 0.05). Therefore, our study suggests that the optimal timing for spraying ZnSO4 on ginkgo is in June, with a recommended concentration of 12 mmol/L. Our study provides a theoretical basis for the accumulation of secondary metabolites in ginkgo and guides the production of its leaf-utilization plantations.