A 12-point checklist in the design and practical application of active surveillance of diseases in aquatic organisms (farmed and wild) has been developed to serve as a methodological approach and guidance for a multidisciplinary team particularly in countries where surveillance expertise is limited. The checklist is based on a review of available main aquatic surveillance references and scientific literature and was further developed based on the outcomes of several aquaculture biosecurity project-related workshops hosted by the Food and Agriculture Organization of the United Nations. The checklist includes the following: (1) scenario setting;(2) defining surveillance objective; (3) defining the populations; (4) disease clustering; (5) case definition; (6) diagnostic testing; (7) study design and sampling; (8) data collection and management; (9) data analysis; (10) validation and quality assurance; (11) human and financial resources and logistics requirements; and (12) surveillance in the bigger picture. For a multidisciplinary approach to disease control, knowledge of fish biology, aquaculture systems and many aspects of aquaculture health management are required. Surveillance needs significant financial investment and must be supported by adequate diagnostic capability, information system management, legal framework and communication networks, with transparent reporting mechanisms to allow rapid disease response for serious diseases of aquatic organisms. It is a stepwise and pragmatic approach that offers a good starting point for addressing disease issues especially in developing countries. It can be used as a model to build targeted surveillance competency and a basic reference when implementing a surveillance programme or improving existing programmes.