An accurate grasp of species distribution is a requirement for biodiversity studies, and understanding species’ biomass is crucial to assessing ecosystem productivity and material cycles. Traditional bioassays and biomass assessments are based on data from trawl surveys. However, trawl surveys often do not provide a true reflection of species distribution and resource status because of differences in the living habits and resource status of different species. To accurately grasp species distribution and resource status, it is imperative to identify cost-effective aquatic organism survey methods to provide technical support for the research and protection of aquatic ecosystem biological diversity. In recent years, the rapid development of molecular biology research has transformed species identification methods from traditional morphological classifications to molecular biology-based techniques, especially the development and application of environmental DNA technology. Environmental DNA is a ubiquitous free radical released from the skin, mucus, saliva, sperm, secretions, eggs, feces, urine, blood, roots, leaves, fruits, pollen, and decaying bodies of DNA molecules. Environmental DNA technology refers to the method of qualitatively or quantitatively analyzing DNA fragments directly from environmental samples (such as soils, sediments, and water bodies) using sequencing techniques. Environmental DNA technology has become a novel method of aquatic organism survey, which is mainly used in processes such as the prevention and control of biological invasion, protection of endangered species, and evaluation of biodiversity and biomass. Here, we review the development of environmental DNA technology, the operational flow, and its application in aquatic ecosystems, as well as the advantages and existing problems. In addition, we project the prospect of environmental DNA in the field of ecology. We hope that this review will provide novel ideas and methods for the research and protection of the biological diversity of aquatic ecosystems to ensure that aquatic resources can be fully utilized.