Recent studies have demonstrated the potential of combining molecular technologies with environmental sampling to detect various vertebrate species in aquatic ecosystems. The European pond turtle (Emys orbicularis) is a threatened and elusive aquatic reptile with shy behaviour. We aimed to develop and evaluate a methodology to detect the presence of this secretive aquatic reptile in ponds from environmental water samples. First, we determined that reptilian DNA can be isolated and amplified from water samples in artificial and natural ponds with known turtle density. Then we compared the potential of two water sampling methods (through filtration or precipitation) and found no significant differences between these approaches. Finally, we demonstrated that the eDNA concentration detected is not correlated with the number of E. orbicularis individuals or biomass. Detection of eDNA was higher in artificial ponds with small volumes of water or in the shallow waters of natural ponds. The eDNA-based methodology aims to detect the presence of specific species, even at low density, with better accuracy than visual observation. However, our study indicates that this method of population monitoring should be applied with caution to aquatic reptiles.