Abstract: Taking the 2-year-old Ginkgo biloba seedlings as test materials, a pot experiment was conducted in an artificial climate chamber to study the effects of air temperature and soil moisture  on the flavonoids accumulation in leaves. Three levels of air temperature (15/5 ℃, 25/15 ℃, and 35/25 ℃ day/night) and three levels of soil moisture  (55%-60%, 40%-45%, and 30%-35% of field capacity) were installed, yielding nine temperaturesoil moisture combinations. Under the three levels of soil moisture, the quercetin, kaempferol, isorhamnetin, and total flavonoids contents in the leaves were higher at 15/5 ℃ than at 25/15 ℃ and 35/25 ℃. Soil moisture  had minor effects on the flavonoids accumulation. The leaf kaempferol content was the highest, followed by quercetin and isorhamnetin. The total flavonoids yield per plant at 35/25 ℃ was higher than that at 15/5 ℃ and 25/15 ℃. It was suggested that to adopt appropriate soil covering and watering before harvesting to decrease the ambient temperature could benefit the enhancement of leaf flavonoids content and the improvement of per unit area flavonoids production in G. biloba leafharvesting plantation.