Abstract: For accurately evaluating the role of agroecosystem in regional carbon balance, the CO₂ flux of typical rice-wheat rotation agroecosystem was continuously observed for a long time by using vorticity correlation technology in Huaihe River Basin. Based on the observation data of CO₂ flux of Shouxian National Climate Observatory from July 2007 to December 2019 after data quality control, the variation characteristics of CO₂ flux in different time scales of rice-wheat rotation agroecosystem in Huaihe River Basin was analyzed. The results showed that there were obvious diurnal, seasonal and interannual variations in CO₂ flux of that in Huaihe River Basin. The diurnal variation of CO₂ flux shows a U-shaped feature, with net absorption in the daytime, maximum at 12:00 BT, and net emission at night, maximum at 21:30 BT. The net absorption in the daytime is significantly higher than the net emission at night. The seasonal variation of CO₂ flux shows a W-shaped double peak feature. The net absorption of CO₂ is from January to May, July to October and December, and reaches the second maximum in April and the maximum in August; the net emission is from June and November. The net CO₂ uptake of wheat and rice at booting stage was -0.207 mg/ (m²·s) and -0.266 mg/(m²·s) respectively. The rice-wheat rotation agroecosystem in Shouxian has strong carbon sequestration capacity, and the annual average net CO₂ absorption from 2007 to 2019 is -2.58 kg/(m²·a). The annual difference is large. In 2008, the net absorption of CO₂ is the largest, -3.26 kg/(m²·a). In 2017, the net absorption of CO₂ is the smallest, only -1.78 kg/(m²·a).During the whole growth period, the total net CO₂ uptake was-2.8 kg/m², accounting for 43.2% of wheat and 56.8% of rice, respectively. The net CO₂ emission of rice-wheat rotation in two intermittent periods was 0.21 kg/m².