The quantification of CO2 flux of water reservoirs is still largely uncertain due to complex temporal variations in physio-chemical parameters and CO2 emissions. To address this issue, the diel-scale characteristics of physio-chemical parameters, δ13CDIC, and CO2 emission flux from Dalongdong reservoir (DLD), a karst groundwater-fed reservoir, in southern China were measured covering July, August, and November of 2014 and March and June of 2015. The results showed that the physio-chemical parameters, δ13CDIC, and CO2 emission flux displayed significant seasonal variations and intricate diel differences in the DLD reservoir. At the seasonal scale, the DLD reservoir is a CO2 source because the mean value of the CO2 emission flux is higher than zero. The highest CO2 emission flux was in November of 2014 due to reservoir overturn and the lowest flux was in August of 2014 due to reservoir thermal stratification. Diel differences in pH, DO, DIC, and pCO2 were inconspicuous during the study periods, while the CO2 emission and δ13CDIC showed higher values during the daytime than the nighttime during July of 2014 and March and June of 2015. In August of 2014, mean differences between the daytime and nighttime CO2 emission were inconspicuous due to a negative CO2 emission flux occurrence. Reservoir overturn in November of 2014 resulted in a higher mean CO2 emission flux and larger data range during the daytime than at nighttime. Future studies that calculate CO2 emissions in water reservoirs should consider monitoring temporal frequencies to obtain more accurate estimations of CO2 emission fluxes.