<p>Environmental DNA (eDNA), the archive of unseen biodiversity, is an emerging and revolutionary biomonitoring approach for detecting species and pathogens in the environment. This approach primarily involves monitoring genetic material shed by microscopic and macroscopic organisms into their environment through feces, mucus, skin cells, and decomposing tissues. Although wetlands are ecologically rich environments, they are highly vulnerable to threats such as habitat degradation, anthropogenic activities, and contamination, leading to decline in water quality and biodiversity loss. In this scenario, eDNA emerges as a promising and non-invasive tool for assessing biodiversity and detecting invasive and elusive species. This review synthesizes recent eDNA research on a global scale, focusing on inland water biodiversity, ecosystem monitoring, and public health interventions. It compares the diverse protocols used across studies, including sampling, pre-treatment, eDNA isolation, amplification, and validation, to highlight existing knowledge and ongoing challenges in this field. This review also outlines the major research gaps, emphasizing the necessity of protocol standardization to enhance the reliability and applicability of eDNA-based research in aquatic environments.</p> Graphical Abstract <p></p>