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Extensive water diversion projects that have been increasingly installed worldwide transport essential water resources as well as a large number of biota. However, studies of the dynamic processes of such transport have been limited. The South-to-North Water Diversion Project of China is the largest manmade water diversion system ever constructed. Here, in a year-long project, we used environmental DNA (eDNA) metabarcoding to assess fish biodiversity and assemblage composition along the Project's 1277-km main canal, while also investigating the temporal, spatial, and functional trait drivers of changes in the fish assemblages. Together, 45 fish taxa were detected, with substantial compositional variations between seasons. The number of detected species typically dropped upon entering the canal but remained relatively constant along the canal's length. Spatial variations in fish assemblages were generally dominated by the turnover component over nestedness, and a positive spatial autocorrelation of qualitative assemblage composition was detected within 80 km in all seasons. Furthermore, several functional traits, such as smaller body size, invertivorous diet, rheophilic living, and lithophilic and demersal spawning, were positive predictors of fish presence along the length of the canal and they may boost species chances of introduction to the recipient areas. Our results provide crucial information for ecological management of diversion projects and have key implications for modelling and predicting foreign species invasion through water transfers. [Display omitted] • Rich fish biodiversity was detected using eDNA along a water-transfer mega-project. • Fish assemblages varied spatially and temporally in the transfer canal. • The turnover component dominated fish assemblage β-diversity along the canal. • Fish biological and ecological traits affect their success of transfer. [ABSTRACT FROM AUTHOR]