【Objective】The massive application of nitrogen fertilizer to agricultural soils plays an important role in ensuring the world’s food supply. However, it also leads to a large amount of reactive nitrogen(N) entering the environment, which strongly interferes with the biogeochemical cycle of N and causes a series of ecological and environmental problems. As the last step of N cycling, denitrification is the predominant pathway, converting reactive N into inert N(i.e., N₂). However, measuring soil N₂ production from denitrification is a major challenge in terrestrial ecosystems because of the high atmospheric background N₂ concentration. Recently, direct methods for measuring N₂emission rates have been developed. Among them, robotized incubation and analyzing system(Robot system) which is based on N₂ free headspace(i.e., helium environment) have been widely used for measuring N₂ emission in pure denitrifying culture or soil, due to its advantage on platform construction and high throughput for N₂ determination. Nevertheless, frequent sampling with the small-diameter steel needle is required during the operation and determination of the Robot system, which inevitably causes leakage of N₂. This seriously interferes with the determination of low N₂ emission rates(i.e., background N₂ flux in upland soil). Therefore, to enable the Robot system to measure background N₂ emission rate in upland soil without exogenous substrate, the leakage rate of the system must be further reduced.【Method】In this study, helium-washed rubber septa, solutions prepared by helium-washed distilled water and destructive sampling treatments were explored to optimize the Robot system aiming at reducing the N₂ leakage therein. Additionally, results of soil N₂ emission determined by the optimized Robot system were compared with those of acetylene inhibition technique(AIT) and Robotized continuous flow incubation system(RoFlow system).【Result】Our results showed that the N₂ leakage rate of the Robot system could be remarkably reduced by optimizing with helium-washed septa, solutions prepared by helium-washed distilled water and destructive sampling treatments. The N₂ leakage rate was reduced to 0~0.78 μL·L^-1·h^-1 by the aforementioned treatments. Under similar treatments, the N₂ emission rate measured by the acetylene inhibition method was highest, followed by the RoFlow system, and the Robot system had the lowest results. Furthermore, the optimized Robot system was capable of determining upland soil N₂ emissions in response to carbon and N addition, which also had the smallest standard error(0.003~0.045 mg·kg^-1·d^-1) compared with the AIT method(0.34~3.29 mg·kg^-1·d^-1) and RoFlow system(0.41~1.02 mg·kg^-1·d^-1).【Conclusion】Overall, the optimized Robot system is characterized by low N₂ leakage, effective response to substrate addition and good consistency in determining soil N₂ emission. In the future, it will have a favorable application in investigating background N₂ emissions and the associated mechanism of upland soil.