[Objective] The ecological network of Natural Reserves in Lancang River basin, Yunnan Province was constructed in order to providing a scientific basis for the ecological security and protection of the basin. [Methods] We constructed a minimum accumulation resistance model of six ecological factors (distance from water, distance from town, vegetation coverage, land use, slope, and elevation). In this model, the city and county-level nature reserves were considered to be the ecological sources. The ecological network of the Lancang River basin was constructed as the least resistance path between different sources, identified as the potential ecological corridor to detect the ecological nodes and ecological fracture points. [Results] A total of 43 ecological sources in the Lancang River basin (mainly in the middle and lower reaches of the basin) were constructed by using the model, with 67 ecological corridors overlapping the main water system of the Lancang River by 59%. Twenty ecological nodes and 41 ecological breakpoints were identified in the ecological network, and these will be used in the optimization and restoration plan of the ecological network and the construction of regional roads in a later period. [Conclusion] According to the distribution characteristics of ecological source areas, ecological corridors, and ecological networks, the number of ecological source areas in the upper reaches of the Lancang River, Weiyuan River basin, and other related areas should be increased. At the same time, the quality of habitats along river banks in the Lancang River basin should also be improved through the use of ecological construction that considers ecological nodes and ecological breakpoints so as to form a complete ecological protection network, alleviate the problem of islanding, and promote the flow of species between ecological sources.