Ginkgo (Ginkgo biloba L.) is widely cultivated for leaves containing flavonoids and ginkgolides with various bioactivities, achieving global sales of preparations exceeding $10 billion. Improving the secondary metabolites contents in Ginkgo leaves holds significant importance for health and socioeconomic aspects. In natural settings, moderate stress boosts the biosynthesis and accumulation of secondary metabolites. However, controlling stress during growth to improve secondary metabolites contents in Ginkgo leaves is impractical due to the multiple, complex, and uncontrollable field conditions. To address the trouble, this study employed exogenous hydrogen peroxide (H2O2), an essential substance that stress affects secondary metabolism, at concentrations of 20, 200, and 2000 mmol·L−1, applied to pre- and post-harvest fresh Ginkgo leaves. Parameters including reactive oxygen species, antioxidant enzymes, osmolytes, flavonoids, and ginkgolides were measured, and metabolomics and bioinformatics were conducted to analyze metabolites and fatty acid desaturase (FAD). As a result, superoxide radical, H2O2, and malondialdehyde significantly increased after treatment with exogenous H2O2, and antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase, as well as osmolytes like proline, soluble protein, and soluble sugar, also notably increased. Concurrently, GbFAD was influenced, including stress-responsive elements, gene expression patterns, protein secondary structure, etc., leading to a significant upregulation of lipids and lipid-like molecules, which helped maintain cell membrane structure and function, facilitating secondary metabolism. Consequently, the secondary metabolites contents in post-harvest fresh Ginkgo leaves appreciably improved, particularly at 200 mmol·L−1, with quercetin, kaempferol, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide increasing by 68.01 %, 26.86 %, 42.99 %, 29.09 %, 39.60 %, 45.83 %, and 51.25 %, respectively. Exogenous H2O2 induces physiological responses in the post-harvest fresh Ginkgo leaves similar to those observed under pre-harvest stress, thereby improving flavonoids and ginkgolides contents. This groundbreaking discovery that exogenous H2O2 can induce stress in post-harvest fresh Ginkgo leaves to increase secondary metabolites contents on an industrial scale not only further advances the Ginkgo-related pharmaceutical industry but also provides a novel approach and perspective for improving secondary metabolites contents in other medicinal crops during post-harvest processing.