This study presents an eDNA sampling protocol optimized for cetacean detection aboard whale watching tours across the Northeast Atlantic Ocean and Mediterranean Sea. By systematically modifying water volume, sampling timing (immediately or delayed after the target animal’s presence), sampling location (from a whale flukeprint or breach site), and filter type, we show that larger sample volumes (10 L), immediate post-encounter collection from either a flukeprint or breach site, and Smith-Root filters (1.2 µm pore size) significantly enhanced cetacean eDNA detectability. A novel order-specific qPCR assay (Cet-CSS) enabled reliable detection of multiple target species across diverse marine environmental samples. These findings support the integration of citizen science into cetacean monitoring efforts and highlight the scalability of eDNA-based approaches for marine research.