In order to investigate the gene expression related to flavonoid biosynthesis in Ginkgo mature leaves, a cDNA library of Ginkgo mature leaves was constructed. 2324 clones were randomly selected for sequencing from the 5' end to construct an EST library, and these sequences were performed homology comparison with nucleic acid database in NCBI by using BLASTX. The result shows that 21 ESTs from 9 genes of Ginkgo mature leaves were highly homological to genes involved in flavonoid biosynthesis from other plant species. Isoflavone synthase gene, chalcone synthase gene and 4-coumarate-CoA ligase gene were expressed in middle abundance, and genes of prephenate dehydratase, phenylalanine ammonialyase, cinnamic acid 4-hydroxylase, 2-hydroxyisoflavanone dehydratase, flavonol synthase and anthocyanidin synthase were expressed in low abundance. This result would offer a scientific foundation for further revealing the gene expression profile of flavonoid biosynthesis in Ginkgo leaves as well as their physiological characteristic, and for further cloning full-length cDNA of above key genes.