Abstract: To clarify the spatial scale effects of land use on the organisms in tropical rainforest streams, this study conducted Yinggeling in Hainan Tropical Rainforest National Park as the research area. Random forest models were used to analyze the spatial scale effects of land use on the biodiversity of stream organisms. The results showed that the explanatory power of land use at different spatial scales for the diversity indices of macroinvertebrate communities ranged from 45.6% to 52.8%. Among them, land use at the watershed scale had an important impact on the Shannon diversity index, and the land use within 3 km upstream of the watershed was an important land use factor explaining the species richness, Simpson diversity index, and evenness index. The explanatory power of land use at different spatial scales for the diversity indices of fish communities ranged from 30.7% to 42.0%. Percent of natural forests and shrub forests within 1 km upstream of the watershed were important land use factors affecting the Shannon diversity index, Simpson diversity index, and evenness index of fish. In conclusion, there are significant spatial scale effects of land use in Yinggeling on macroinvertebrate and fish biological communities, and the stream biological communities in this area are most sensitive to the land use within 1 km and 3 km upstream of the watershed. The research results provide data supports and theoretical basis for the land use planning and protection of stream biodiversity in the Yinggeling area of Hainan Tropical Rainforest National Park.