The assessment of the occurrence of rare and endangered species in freshwater environments is crucial for ecological studies and conservation issues, but it can be time-consuming and challenging in harsh environments. Detecting DNA traces from the environment (environmental DNA, eDNA) can provide innovative and reliable solutions for the monitoring and conservation of rare and elusive species, such as the Eurasian otter Lutra lutra. We tested an experimental workflow based on target qPCR assay to detect L. lutra eDNA from water samples as a speditive monitoring tool at large scale to be coupled with fine-scale traditional field surveys. This is the first application of an eDNA-based approach to monitor the presence of L. lutra in Italy. We compared the eDNA-based results with traditional survey observations and confirmed the reliability of this innovative approach for the large-scale monitoring of such aquatic elusive species.