Abstract: To clarify the spatial scale effects of land use on the organisms in tropical rainforest streams, we selected the Yinggeling in Hainan Tropical Rainforest National Park as research area and used Random Forest model to analyze the spatial scale effects of land use on the biodiversity of stream organisms. The results indicated that the explanatory power of land use at different spatial scales for the diversity indices of the benthic macroinvertebrate communities ranged from 45.6% to 52.8%. Among them, the land use at the watershed scale had an important impact on the Shannon diversity index, and the land use within 3.0 km of the watershed upstream was an important land use factor which can explain significant variations of species richness, Simpson diversity index, and evenness index. The explanatory power of land use at different spatial scales for the diversity indices of the fish communities ranged from 30.7% to 42.0%, and the natural forests and the shrub forests within 1.0 km of the watershed upstream were the important land use factors which can explain significant variations of Shannon diversity index, Simpson diversity index, and evenness index of fish. In conclusion, there were significant spatial scale effects of land use in the Yinggeling on the benthic macroinvertebrate communities and the fish communities, and which are most sensitive to the land use within 1.0 km and 3.0 km of the watershed upstream. Our findings provide the data support and the theoretical basis for the land use planning and protection of the stream biodiversity in the Yinggeling of Hainan Tropical Rainforest National Park.