Abstract: Chinese sturgeon (Acipenser sinensis) is a flagship species in the Yangtze River basin. Under the background of the 10-year fishing ban in the Yangtze River, it is of great significance to study the nondestructive testing technology of Chinese sturgeon. Environmental DNA (eDNA) technology is an environmentally friendly biological monitoring technology, which can detect species without direct observation or capture of the organism. It was tried to screen out the specific primers that could be used to detect the eDNA of Chinese sturgeon from the literature. In September 2020, four areas where Chinese sturgeon often appeared were selected in the middle and lower reaches of the Yangtze River, and three-dimensional sampling was performed on each section. eDNA of 16 sites was extracted, and the screened primers were used to detect the eDNA of Chinese sturgeon, in order to try to explore the distribution characteristics of Chinese sturgeon. A group of primers that could detect the eDNA of Chinese sturgeon was successfully screened. By using this primer, the eDNA of 4 species of sturgeon in the Yangtze River including the Chinese sturgeon was successfully detected, and about 3 million sequences of sturgeon were measured. Based on the sequencing results, the differences in the eDNA of Chinese sturgeon detected in different sections were analyzed. It was found that the eDNA in Yichang River section was the most, and the eDNA in Dongting Lake mouth section was the least, and there were significant differences in the eDNA detection between the surface and bottom waters. The study found that the screened primers could be used for the detection of eDNA of Chinese sturgeon, and the detection results of eDNA were consistent with the historical investigation and migration characteristics of Chinese sturgeon. There were significant differences in the amount of Chinese sturgeon eDNA detected under different water depths, which indicated that mixed or three-dimensional sampling may be more comprehensive for the detection of Chinese sturgeon eDNA in the future investigation.