Macrozoobenthos are the most diversified organisms in freshwater ecosystem, playing a vital role in maintaining the structural and functional integrity of freshwater ecosystem. They are one of the most significant bioindicators that have been widely used in water quality monitoring and assessment. Nowadays, anthropogenic disturbance, pollution and global warming lead to a remarkable decline of the macrozoobenthos biodiversity. Therefore, how to provide substantial data for macrozoobenthos biodiversity monitoring rapidly, accurately, and reliably has become a major issue in water environment management. However, the traditional morphology-based biomonitoring survey method can hardly fulfill current biomonitoring requirements due to being costly, time consuming, and labor intensive. Moreover, such method is highly dependent on experienced taxonomists and often results in low precision and unverifiable taxonomic data. Environmental DNA metabarcoding (eDNA metabarcoding), a newly emerged approach, is considered as a promising survey tool for aquatic biodiversity monitoring. Compared with traditional biomonitoring survey methods, eDNA metabarcoding remarkably outperforms morphology-based method by its low cost, straightforward workflow, and high sensitivity in species detection of macrozoobenthos. Particularly, it circumvents the taxonomy literature and expertise, providing rapid, accurate, and high-throughput identification simultaneously to a large variety of taxa regardless of the organism conditions and environmental disturbances, thus permitting the easy access of large scale biomonitoring programs for freshwater ecosystem. Currently, the application of eDNA metabarcoding in macrozoobenthos biodiversity monitoring is largely affected by several technical factors, such as the sampling protocols, primer selection, and the completeness of reference database. Particularly, different methods and procedures often lead to contrasting results. Hence much attention is drawn to the field workflow and laboratory methodologies to be standardized to allow results to be compared across studies. In this paper, we reviewed the recent published studies referring to macrozoobenthos biodiversity monitoring using eDNA metabarcoding to discuss its definition, advantages, applications and its methodologies used in macrozoobenthos biodiversity biomonitoring and assessment. Especially we discussed the vital technical factors that influences eDNA metabarcoding, including sample collection protocol, gene marker selection, primer design, PCR bias, the completeness of reference database, and their corresponding recommendations to be optimized. Based on the summary above, we give several helpful suggestions for improving the efficiency and accuracy of macrozoobenthos monitoring using eDNA metabarcoding. We expect that this review will provide reliable guidelines for developing a sound macrozoobenthos biomonitoring design using eDNA metabarcoding. At last, we gave perspectives regarding several emerging applications and innovative methodologies of eDNA metabarcoding can be used in ecology.