As a compensation for the loss of fish habitat caused by the development of high-dam hydropower in the mainstream, tributary habitat substitution has been widely used to protect native fish in the river affected by the projects. However, what kind of protection effect of substitution tributary plays and the impact of the mainstream on it remain unclear. In this work, the Jidu River and Luosuo River were selected as the research objects, and the neighboring tributaries were selected as the control groups. Through analysis and comparison of the fish survey data, the differences of fish species and community structures in each tributary were revealed, and the effects of tributary habitat substitution and the influence of the mainstream on it were investigated. Results showed that the tributary habitat substitution had obvious protection effects on the mainstream fish. The fish species richness was significantly improved after the restoration of the tributary habitats. The number of native fish and endemic fish species increased by 12 and 7 respectively in the Luosuo River, while the corresponding increments were 7 and 2 species in the Jidu River. The tributary habitat substitution could provide the key habitat for the native fishes, some endangered endemic fishes, and migratory fishes of Lancang River to complete their life history. The mainstream had important influences on the substitution tributaries. Bray-Curtis similarity of fish communities in Luosuo River and Jidu River were 21.76% and 10.73%, respectively. Maintaining the "riverine zone" in the tributary estuary was the key to the sustainable protection. Considering the benefit and investment of tributary habitat substitution, it is recommended that the tributaries in the reservoir tail should be given priority when carrying out such protection measures in the future.