Using a grid-based sampling method, 2755 soil profiles consisting of 23536 soil samples were sampled and analyzed to study the spatial distribution of soil organic carbon (SOC) in the karst basin of Guizhou Province. Further, we established an estimation method of SOC stock exclusively for karst after correcting for soil distribution area, particle content, percentage of exposed rock, and soil thickness, and optimizing the calculation formula. The results showed that there were obvious differences in the SOC content of different soil types and at different soil depths. The content of SOC decreased with an increase in soil depth, but the extent of the decrease differed in soils of different types. The content of SOC in soils on shady slopes was higher than that in soils on sunny slopes. The content of SOC at different slope positions decreased in the following order:upper-middle, top, middle, lower-middle, and bottom. There was extensive SOC heterogeneity in the karst area. The order of SOC content under different land use patterns was as follows:woodland > shrub grassland > dry land > paddy field. SOC content showed a very significant positive correlation with slope, elevation, and percentage of exposed rock, and a significant negative correlation with soil thickness and soil bulk density. There was a large spatial variability in SOC in the karst area, with the influences of different indicators for SOC reserve estimation being as follows:soil thickness > percentage of exposed rock > particle content > content of SOC > soil bulk density. Using the revised formula to make estimations, the SOC density of the surface 20 cm of soil in a small watershed of the Houzhai River catchment in Pudin was 3.53-5.44 kg/m², with an average value of 1.24 kg/m². The SOC of the surface 100 cm of soil was 4.44-14.50 kg/m², with an average value of 12.12 kg/m², and the SOC storage was 5.39×10⁵ t.