Biochar (BC) is widely used in soil to increase carbon sequestration. However, the long-term effects of BC application on the soil organic carbon (SOC) and labile organic carbon (LOC) fractions are unclear, especially in karst regions. In order to address this issue, two field experiments were designed to observe the changes in the SOC and its fractions after two years of BC application. In this study, we chose karst regions with two different ecological types, Linquan and Heishitou towns in Bijie City, China. Five BC treatment plots were established, with application rates of 0, 5, 15, 20, and 40 t center dot ha(-1) (CK, B-5, B-15, B-20, and B-40, respectively). Compared with CK, the contents of SOC, readily oxidizable organic carbon (ROC), particulate organic carbon (POC) and recalcitrant carbon (RC) under the BC treatments in Linquan increased by 7.09-38.08%, 1.62-39.80%, 9.52-62.30% and 8.66-58.14%, respectively. In Heishitou, the increases in these contents were 3.60-38.58%, 4.40-115.23%, 19.57-50.98% and 10.94-53.87%, respectively. There are significant positive relationships between the SOC content and the ROC, POC and RC contents. However, the application of BC had no significant effect on the DOC content in the two ecological regions. In addition, the soil carbon pool index (CPI) and carbon pool management index (CPMI) increased with increasing BC application rate due to its unique physicochemical properties and nutrients. These results indicate that long-term BC amendment significantly improves the soil quality and carbon sequestration in karst regions by increasing SOC and its LOC fractions.