Thermal stratification and mixing are important to the physicochemical compositions of reservoirs and lakes that will impact on water quality and biogeochemical cycles. However, it remains unclear how thermal stratification and mixing process control exchange of CO2 between surface water and atmosphere. To address this issue, we examine the temporal characteristics of some physicochemical parameters, partial pressure of CO2 (pCO2), the δ13CDIC, and the CO2 emission flux in a typical karst groundwater-fed reservoir (Dalongdong reservoir) for 18 months in 2016–2018. Our results demonstrate that thermal stratification plays an important role in limiting CO2 emission from early April to late October, while mixing processes stimulates more CO2 emission from October to April. The Dalongdong reservoir is a source atmospheric CO2 for most of the study period; however, significant temporal variations of CO2 emission appeared due stratification and breakdown of stratification. During periods of stratification, CO2 fluxes were into the water with approximately -845.46 t CO2/y captured, while approximately 14062.58 t CO2/y were lost to the atmosphere during periods lacking stratification. This study information that could help optimize sampling monitoring strategies to reduce errors of regional CO2 emission estimation.