Environmental DNA (eDNA) analysis is considered a powerful tool for estimating the biomass as well as the presence / absence of aquatic organisms in rivers. However, few studies have clarified the relationship between biomass and environmental DNA concentration in running water experiments. In this study, we revealed the relationship between eDNA concentration and biomass of Dark chub Candidia temminckii in a running water experiment. Furthermore, we attempted to quantify the eDNA concentration of C. temminckii at three rivers in Chugoku district, Japan. The results of the running water experiment revealed a clear positive relationship between the eDNA concentration and the total weight of C. temminckii. In addition, the eDNA of C. temminckii was detected in samples from all sites.