In order to investigate the ecological instream flow and ensure the water security and ecological environment health of the river basin, the proposed analysis was carried out on the ecological instream flow of Kaidu River by four hydrological methods including the Tennant method, the typical hydrological year method, the driest month average flow method and Qp method based on the measured runoff data of the Dashankou Hydrological Station from 1960 to 2018. Comparative analysis suggests that the calculation result of Tennant method is the most suitable. The average ecological instream flow of Kaidu River at Dashankou Hydrological Station from October to March of the following year should be no less than 15.60 m3/s, and the corresponding minimum ecological environmental water requirement is 2.47×108 m3. Whereas the average ecological instream flow from April to September should be no less than 46.15 m3/s, and the corresponding minimum ecological environmental water requirement is 7.30×108 m3. Furthermore, the average annual ecological instream flow is no less than 30.88 m3/s, and the corresponding minimum ecological environmental water demand is 9.77×108 m3. It is found that these objectives of ecological instream flow can make up for the water loss of the section between the mountain and Bosten Lake of Kaidu River, ensure the basic ecological function of the river and the security of water ecology, as well as provide support for the ecological instream flow management of the river.