Species distribution and biomass are the basis for evaluating population dynamics and community structure in an ecosystem. Unfortunately, it is frequently full of challenges to capture the distribution of the rare species through traditional methods. Procypris rabaudi is a unique economic species in the upper reaches of the Yangtze River, the number of which has declined dramatically in recent years. Environmental DNA technology is a sensitive, low-cost and non-invasive new technology for species monitoring. It has great potential application in detecting endangered and invasive species. In order to establish a real-time quantitative PCR (qPCR) method for the detection of P. rabaudi and distinguish it from other fishes in the Yangtze River, this study designed eDNA primers and a TaqMan probe based on 12S rRNA gene sequence in mtDNA. The sequence of the 12S rRNA gene was amplified by PCR and cloned into the pMD19-T vector to construct the standard plasmid, and a qPCR method was developed for detection of P. rabaudi using serially diluted standard plasmid as templates. Subsequently the sensitivity, specificity and application effects of the method were evaluated. The results showed that the cycle threshold value (C_t) of qPCR assay had a great linear relationship with the copy number of the standard plasmid. Amplification specificity analysis indicated that the method could specifically detect P. rabaudi. Then, eDNA was detected in the water samples in aquarium tanks with different numbers of P. rabaudi, The target DNA concentration and the number of P. rabaudi had a positive correlation of R² was 0.957, and the correlation curve between DNA concentration and the individual number was obtained: y = 131 546x + 77 623. In addition, after the removal of P. rabaudi, the copies of eDNA were negatively correlated with time, and its retention time in the water environment was about 17 days. In this study, we found that the DNA primer and Taqman probe could be applied to the qualitative detection of P. rabaudi in water with high specificity, as well as estimate fish density in tanks quantitatively. These results demonstrate that eDNA analysis method is a potential tool to reflect the biomass of P. rabaudi in different sampling sites, which can provide a basis for assessment of artificial release effect and resource management of P. rabaudi in the future.