Background: Ginkgo is a native plant to China which is recently cultivated in Iran. The extract of the Ginkgo leaves contains flavonoids and ginkgolids and has been used pharmaceutically for a while. Flavonoids of Ginkgo have been shown to have several effects which could increase blood flow of brain and limit the damage associated with ischemia also they inhibit cyclo-oxygenases and lipoxygenases activities and show antioxidant effects. The researchers showed that Ginkgo leaves flavonol glycosides varies during different seasons. Objective: The aim of the present research is study on seasonal variation of flavonoids of Ginkgo biloba leave’s cultivated in Iran by HPLC method. Methods: In this study Ginkgo leaves from 5 years old trees collected in 2005 from North of Iran and evaluated. Plant leaves were dried, milled, and extracted with a suitable solvent. For analysis of flavonoid aglycones in the extract, flavonol glycosides were hydrolyzed in acid medium and identified by HPLC instrument and the peaks of standard material Quercetin, Kaempferol and Isorhamnetin were obtained. Results: According to our findings, flavonoids vary in amount during seasons and upper most amount was found in June Month (5.5%w/w). Conclusion: Results of our study shows a mild variation of flavonoids content in Ginkgo tree leaves during different months, also we found a high concentration of flavonoids in young tree leaves that is very higher than pharmacopeia′s standard (0.5% w/w) and these leaves are suitable for preparing of standardized extracts.