The flavonoids content and its composition in Ginkgo (Ginkgo biloba L.) leaves are affected by environmental factors such as temperature and soil moisture. Here we performed experiments in phytotron using 2-year-old Ginkgo seedlings to explore the effects of temperature and soil moisture on flavonoids content, enzymes related to flavonoids biosynthesis such as phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and p-coumarate CoA ligase (4CL), soluble sugar and protein content. We found that temperature and lower soil moisture had significant effects on these parameters. The effects of temperature on flavonoids content, the activity of C4H and 4CL and soluble sugar content were greater than that of soil moisture. The total flavonoid content, the activity of PAL, C4H and 4CL, soluble sugar content were higher in lower temperature regime 15 A degrees C (daytime)/5 A degrees C (nighttime) and lower soil moisture (30-35 % of field capacity), but higher temperature was beneficial to the accumulation of soluble protein. This indicates that increasing of soluble sugar content and the activity of PAL, C4H and 4CL are beneficial to flavonoids biosynthesis and accumulation in the Ginkgo leaves, while increasing of soluble protein is adverse to flavonoids biosynthesis. Because lower temperature and soil moisture are favorable to flavonoids biosynthesis, we can take some silvicultural steps to increase flavonoids production in Ginkgo plantation, such as establishing leaf-harvest plantation at lower temperature zone, reducing irrigating before harvesting leaves.