The Yangtze River Basin, the world's third-largest river basin and a hot spot for global biodiversity conservation, is facing biodiversity crisis caused by reduced river connectivity. The deterioration arises from four dimensions: longitudinal, lateral, vertical and temporal. However, limited research has quantified the spatiotemporal connectivity of the Yangtze River Basin and further evaluate the consequent impact on fish biodiversity. In our study, a multi-index evaluation framework was developed to assess the variations in the four-dimensional connectivity of the Yangtze River Basin from 1980 to 2020, and fish biodiversity affected by reduced connectivity was detected by eDNA metabarcoding. Our results showed that the Yangtze River Basin suffers from a pronounced connectivity reduction, with 67% of assessed rivers experiencing deteriorated connectivity in recent years. The lost fish biodiversity along the river reaches with the worst connectivity was likely attributed to the construction of hydropower plants. The headwaters and the down streams of most hydropower plants had a higher fish biodiversity compared with reservoirs. The free-flowing reaches in the downstream of the lowest hydropower station, had higher lotic fish abundance compared with that in the upstream. As for the entire Yangtze River Basin, 67% of threatened fish species, with 70% endemic species, were threatened by reduced river connectivity. Our result indicates that the massive loss of river connectivity changes the spatiotemporal patterns of fish community and threatens protected fish. More effective measures to restore the populations of affected fish in rivers with reduced river connectivity are required.