Biological soil crusts (biocrusts), which develop commonly in the southwestern karst regions, have a significant effect on reducing soil erosion. To explore the effect of biocrusts on soil anti-erodibility, biocrusts with different rehabilitation ages (0, 3, 5, 8, 11 a) were studied on a karst hillslope in Guizhou, and different levels of flow discharge (0.2, 0.3, 0.4, 0.5 L/s) and slope gradients (5°, 12°, 17°, 23°) were designed. The results showed that:(1) Biocrusts on karst hillslopes could promote soil particles cementation, strengthen soil aggregate stability, improve soil structure, and enhance soil water holding capacity and permeability. (2) The biocrusts greatly enhanced the anti-disintegration and anti-shear strength of soil. Compared with the treatment of removing biocrusts, the soil's anti-disintegration and anti-shear strength of the treatment of retained biocrusts increased by 24.83%~46.62% and 25.77%~37.73%, respectively. (3) The tensile strength of the biocrust layer ranged from 1.95 to 5.76 N during the rehabilitation ages (3~11 a). As the development age of biocrusts increased, the structure of the biocrust layer became more stable, and its ability to resist damage became stronger. (4) The biocrusts could also significantly improve the soil anti-scour capacity, which was regulated by flow discharge and slope gradient. The critical value for flow discharge and slope gradient was 0.4 L/s and 17°, respectively, beyond which the protective effect of biocrusts was significantly weakened. These results were of great significance for accurately estimating soil erosion and correctly evaluating ecological restoration construction in karst regions.