Runoff generation is an important part of water retention service, and also plays an important role on soil and water retention. Under the background of the ecosystem degradation, which was caused by the vulnerable karst ecosystem combined with human activity, it is necessary to understand the spatial pattern and impact factors of runoff generation in the karst region. The typical karst peak-cluster depression basin was selected as the study area. And the calibrated and verified Soil and Water Assessment Tool (SWAT) was the main techniques to simulate the runoff generation in the typical karst basin. Further, the spatial variability of total/surface/groundwater runoff was analyzed along with the methods of gradient analysis and local regression. Results indicated that the law of spatial difference was obvious, and the total runoff coefficients were 70.0%. The groundwater runoff was rich, about 2–3 times the surface runoff. Terrain is a significant factor contributing to macroscopic control effect on the runoff service, where the total and groundwater runoff increased significantly with the rising elevation and slope. The distribution characteristics of vegetation have great effects on surface runoff. There were spatial differences between the forest land in the upstream and orchard land in the downstream, in turn the surface runoff presented a turning point due to the influence of vegetation. Moreover, the results of spatial overlay analysis showed that the highest value of total and groundwater runoff was distributed in the forest land. It is not only owing to the stronger soil water retention capacity of forest ecosystem, and geologic feature of rapid infiltration in this region, but also reflected the combining effects on the land cover types and topographical features. Overall, this study will promote the development and innovation of ecosystem services fields in the karst region, and further provide a theoretical foundation for ecosystem restoration and reconstruction.