The Yangtze finless porpoise, (YFP) is the flagship of aquatic protection species in the Yangtze River in China. The number of Yangtze finless porpoises is continuously decreasing because their living conditions are affected by human factors. Determining the distribution of the finless porpoises is necessary to protect them; however, surveys all along the Yangtze River are time-consuming and expensive. Environmental DNA (eDNA) technology used for the detection of organisms is economical, efficient, accurate, sensitive, and non-invasive and can narrow the scope of investigation, which allows limited conservation funds to be used more efficiently. It has already been applied to detect and estimate the distribution of endangered or invasive aquatic species. Environmental DNA technology often deals with a small amount of degraded and short DNA fragments, which implies that maximizing the eDNA yield is required to improve the accurate detection of the target organism from turbid stream systems. In this study, we compared the effects of four different pore-sized filters (10 μm, 5 μm, 1 μm, 0.45 μm) on the filtration time of eDNA during its extraction from stream water. Also, we compared the eDNA yield from water samples stored under two different conditions (room temperature 20℃, refrigerated 4℃) over a 14 day period. Results showed that the target eDNA was detected from the four different pore-sized filters; however, the filtration time was negatively correlated with the pore sizes of the filters. Considering the filtration time, the combination of a large pore-sized filter (5 μm) and a high volume of water (1 L) from the samples is a good choice to extract eDNA from the Yangtze River. Different storage methods and times significantly affected the eDNA yield. Thus, our results also support the recommendation that the water samples from the Yangtze River should be filtered within 6 hours of collection at room temperature or be stored at 4℃ for a short period of time (i.e., 48 hours). The eDNA concentration in the water samples dropped below the threshold for detection when they were stored at room temperature for 10 days or at 4℃ for 14 days; thus, the eDNA in the water samples is not suited for long-term preservation. Compared with traditional field investigations, the eDNA survey exhibited a 100% detection rate from the Yangtze river in the Jiangsu Province where the finless porpoise has emerged. Three positive results were also detected from 10 survey points where the finless porpoise has not yet been observed. The fitted curve between the number of finless porpoises and the eDNA yield was built to estimate the biomass of the porpoises within an area of water. Although eDNA production and degradation is affected by many environmental factors such as temperature, pH, and microbial communities, the technology was still more sensitive than traditional field investigations for the detection of the Yangtze finless porpoise in the Yangtze River.