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The efficient maintenance of the activity of excised branches is the powerful guarantee to accurately determine gas exchange flux between the detached branches of tall trees and the atmosphere. In this study, the net photosynthetic rate (NPR) of the excised branches and branches in situ were measured simultaneously by using two photosynthetic instruments to characterize the activity of the excised branches of Phyllostachys nigra. The ratio of normalized NPR of excised branches to NPR in situ was used to assess the photosynthetic activity of detached branches. Based on photosynthetic activity, an optimal hydroponics protocol for maintaining activity of excised P. nigra branches was presented: 1/8 times the concentration of Gamborg B5 vitamin mixture with pH = 6. Under the best cultivation protocol, photosynthetic activity of excised P. nigra branches could be maintained more than 90％ within 6 hr in the light intensity range of 200-2000 μmol/(m2·sec) and temperature range of 13.4-28.7°C. The nitrogen dioxide (NO2) flux differences between in situ and in vitro branches and the atmosphere were compared using double dynamic chambers. Based on the maintenance method of excised branches, the NO2 exchange flux between the excised P. nigra branches and the atmosphere (from ?1.01 to ?2.72 nmol/(m2·sec) was basically consistent with between the branches in situ and the atmosphere (from ?1.12 to ?3.16 nmol/(m2 sec))within 6 hr. Therefore, this study provided a feasible protocol for in vitro measurement of gas exchange between tall trees and the atmosphere for a period of time.