In order to explore the vertical distribution characteristics and influence mechanism of organic carbon in the deep water reservoir, taking the southwest karst hinterland reservoir (Pingzhai Reservoir) as case study, water samples were collected at different points during thermodynamic stratification and mixing periods, using mathematical statistics, correlation analysis, relative water column stability (RWCS), to analyze the influence of thermal conditions and hydrodynamic differences on the spatiotemporal and cross-sectional distribution on total organic carbon (TOC). The results show that the thermocline is present in spring, summer and autumn, and the thermocline thickness varies by season. TOC concentration is 0.44–5.65 mg/L, and the annual mean value is 2.16±0.99 mg/L. The seasonal variation pattern of TOC concentration is summer>winter>spring>autumn. There is the seasonal difference of the response between TOC and water environment in Pingzhai Reservoir. Exogenous carbon storage in summer, autumn rainfall dilution, microbial decomposition and sediment release in winter, and algae propagation, biological carbon pump effect in spring are the main causes of the seasonal change of TOC concentration. The fluctuation amplitude of TOC and physicochemical factor profiles in different deep water points are mainly driven by three different hydraulic characteristics. RWCS mainly affects the sedimentation rate of substances such as organic carbon, which results in the difference of TOC concentration and physicochemical factor profile distribution.