We used environmental DNA metabarcoding to assess fish biodiversity and assemblage composition along the the main canal of South-to-North Water Diversion Project over a year and to investigate 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 length of the canal. 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, lithophilic and demersal spawning, were positive predictors of fish presence along the length of the canal and thus 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.