Freshwater and estuarine ecosystems support diverse fish communities and provide essential ecological, cultural, and socioeconomic values. Monitoring fish presence and biodiversity across these systems is fundamental for assessing ecosystem conditions, detecting environmental change, and informing conservation and management actions. Environmental DNA (eDNA) has emerged as an efficient, non-invasive approach for detecting aquatic biodiversity, enabling broad-scale monitoring of fish communities across a range of environments. Its application has also created new opportunities for collaborative monitoring, with increasing emphasis placed on integrating local ecological knowledge from community groups and waterway users to improve sampling design and support long-term monitoring. In this context, this project integrates local knowledge of fishing groups, project partners, and collaborators within the sampling design, guiding site selection while establishing a collaborative framework for long-term eDNA monitoring of freshwater and estuarine fish biodiversity in the Wet and Dry Tropics of Tropical Northern Queensland. Specifically, the study combines local ecological knowledge with a transparent and reproducible Multi-Criteria Decision Analysis (MCDA) framework, in which candidate sites are evaluated against six assessment criteria weighted using the Analytic Hierarchy Process (AHP). Knowledge on fish occurrence and waterway condition, is incorporated through participatory workshops where community members and waterway users identify priority waterways, culturally significant locations and site-specific considerations that directly inform the site selection process.