AbstractOver the last years, the analysis of environmental DNA (eDNA) has become an important approach to investigate species communities. Due to its non‐invasive nature and high sensitivity, this method is excellently suited to monitor the occurrence of endangered or invasive species. Native freshwater crayfish are threatened across Europe. Due to their nocturnal activity and hidden lifestyle, crayfish monitoring is difficult and time consuming with conventional methods. Thus, eDNA‐based monitoring of native, as well as invasive crayfish, would be an immense advantage in the conservation of these animals. However, although several assays for the detection of various crayfish species have been published, comparably little is known on the relationship between eDNA concentration and crayfish population densities; a pre‐requisite for estimating population sizes from eDNA measurements. In this study we, therefore, performed a set of laboratory experiments to investigate the relationship between eDNA concentrations and crayfish densities—measured as either crayfish size or biomass. We found a strong correlation between the two measurements and could also show that the amount of eDNA showed a linear increase during the first three days (at least) after crayfish were introduced to aquaria. Additionally, our experiments also suggest that crayfish activity might have a strong influence on the eDNA signal strength. Our findings will help to optimize eDNA‐based monitoring of freshwater crayfish, important for the conservation of these charismatic animals.