The hydrological cycle process in karst landscapes is significantly different from other regions, and the water supply service of the karst ecosystem is also unique. However, the current studies on the spatial and temporal variation of water yield in karst areas seldom take into account the special characteristics of karst ecosystems, resulting in some biases in the spatial simulation results of water yield. In this article, a water yield partitioning method was proposed based on the characteristics of karst landscape, and a conceptual model for water yield correction was established by combining the water balance method and groundwater recharge and discharge characteristics. We selected the Li River Basin, a typical karst area, as the study area, calibrated the water yield simulation results of the InVEST model by the above method, and compared the spatial and temporal distribution patterns of water yield in the Li River Basin before and after calibration from 2000 to 2020 at three scales:raster cell, karst geomorphic unit, and sub-basin. The results showed that:(1) water yield simulated with the InVEST with a spatial expression of model in the study area presented a trend of decreasing, increasing, and then decreasing from 2000 to 2020, high in the north and low in the south. Water yield hardly differed between the karst and non-karst areas, but varied more within the karst areas. Specifically, the water yield decreased in the order of bare, covered, and buried karst with the exposure of water-bearing rock formations and in the order of plain, depression, and peak-cluster with the change of geomorphology. The water yield decreased in the order of upstream region of Li River, midstream region of Li River, Gongcheng River region, Lipu River region and downstream region of Li River. (2) The water supply services corrected with the water yield partitioning method were higher in the northwest than that in the southeast, and higher in the surrounding than that in the middle. The water yield showed significant differences between karst and non-karst areas, and also within different karst units. Specifically, the water yield decreased in the order of buried, covered, and bare karst with the exposure of water-bearing rock formations and in the order of plain, peak-cluster, and depression with the change of geomorphology. The water yield decreased in the order of midstream region of Li River, upstream region of Li River, Gongcheng River region, Lipu River region and downstream region of Li River. (3) Compared with the results of the InVEST model, the water yield corrected with the water yield partitioning method was closer to the measured runoff values, and the spatial and temporal distribution of corrected water yield was more accurate in quantitatively assessing the available water resources in the karst region. This study can provide a scientific and reasonable data basis for water resources protection decision-making and ecological compensation mechanism establishment.