The nocturnal boundary layer (NBL) significantly influences the dispersion and fate of atmospheric species at night. Subtropical forests are crucial in carbon and water exchange between the biosphere and the atmosphere. However, the NBL characteristics and their impact on atmospheric species over these forests remain unknown. This study conducted vertical measurements of atmospheric species such as O₃ and volatile organic compounds (VOCs), along with meteorological variables, over a national forest reserve in Southern China. Results reveal that the NBL height ranged from 180 to 300 m in the summer and from 80 to 160 m in the winter. The vertical distribution of chemical species varied by time and season, with greater concentration gradients observed in the summer. Over 90% of VOCs above the NBL were anthropogenic, while biogenic VOCs were mainly found within the NBL. Higher O₃ concentration and VOC product-to-reactant ratios were observed in the residual layer, suggesting enhanced oxidation levels. This unique vertical distribution of atmospheric species at night is driven by factors, such as emission, deposition, turbulence, and atmospheric chemistry, potentially affecting ecosystem functions. Results from this study highlight the importance of incorporating NBL dynamics into atmospheric models to better understand the evolution of chemical species and their ecological effects over forests.