In this study, using the, Illumina HiSeq high-throughput sequencing technology, we investigated the diversity and structure of endophytic fungal communities harbored in the Ginkgo leaves collected from nine(three groups) ancient Ginkgo with different ages from Hupingshan National Nature Reserve, Hunan Province. The result showed that the endophytic fung diversity, structure and composition were significantly different. The differences of endophytic fungal structure increased with the ages of the studied Ginkgo. Ascomycota was the dominant phyla and Aspergillus, Candida, Mycosphaerella were the dominant genus.The determination of flavonoids in Ginkgo leaves found that the contents of three flavonol glycosides(quercetin, kaempferol and isorhamnetin)decreased when the growth years increased. It concluded that, as an important environmental factor, the flavonoids in Ginkgo leaves had a significant effect on the endophytic fungal communities possibly through impacting the colonization and growth of the dominant Ascomycota genera.