Forests play a crucial role in carbon cycling, contributing significantly to global carbon cycling and climate change mitigation, but their capture strength is sensitive to the climatic zone in which they operate and its adjoining environmental stressors. This research investigated the carbon dynamics of a typical deciduous forest, the Daniel Boone National Forest (DBNF), in the Mixed-Humid climate of Kentucky, USA, employing the Eddy Covariance technique to quantify temporal CO2 exchanges from 2016 to 2020 and to assess its controlling biometeorological factors. The study revealed that the DBNF functioned as a carbon sink, sequestering -1515 g C m-2 in the study period, with a mean annual Net Ecosystem Exchange (NEE) of -303 g C m-2yr-1. It exhibited distinct seasonal and daily patterns influenced by ambient sunlight and air temperature. Winter months had the lowest rate of CO2 uptake (0.0699 g C m-2 h-1), while summer was the most productive (-0.214 g C m-2 h-1). Diurnally, carbon uptake peaked past midday and remained a sink overnight, albeit negligibly so. Light and temperature response curves revealed their controlling effect on the DBNF trees' photosynthesis and respiration. Furthermore, clear seasonality patterns were observed in the control of environmental variables. The DBNF is a carbon sink consistent with other North American deciduous forests.