Enhancing early detection and rapid response (EDRR) of biological threats is critical to prevent further ecosystem damage from established and emerging invasive species. Environmental DNA sampling provides key capabilities for EDRR, as well as long-term monitoring following management or eradication efforts. Automated eDNA samplers (robots) that can be distributed across space and obtain high frequency samples over time have the potential to serve as efficient and sensitive early detection and monitoring tools. In coordination with the United States Department of Interior National EDRR Framework, we are building the Rapid eDNA Assessment and Deployment Initiative & Network (READI-Net), led by U.S. Geological Survey scientists and the Monterey Bay Aquarium Research Institute (MBARI). The broad goals of this project are to enhance the ability of resource managers to develop EDRR strategies for aquatic biological threats early in the invasion timeline. Outcomes include the development and deployment of eDNA robots in pilot surveillance networks, standard operating procedures and quality management to ensure confidence in eDNA results and inferences, along with a laboratory information management system to deliver eDNA detection information to decision-makers. To ensure data harmonization across different labs and timepoints, initial minimum best practices and data standards were developed through broad eDNA community engagement that will be leveraged by READI-Net. These best practices include data and metadata standards for the field, lab, and analysis and interpretation steps and experimental controls, analytical procedures, and thorough validation. To inform network design, sample collection at various spatio-temporal frequencies is informing model-optimization tools for efficient deployment of eDNA robots. Additionally, a communication framework has been established to engage partners and managers about eDNA sample collections and detections within their jurisdictions. Future directions could leverage this network for community wide bioassessment or biodiversity surveys, habitat restoration efforts, and in situ analyses providing remote sensing capabilities.