The mega river ecosystem of the Yangtze River was once home to diverse aquatic megafauna but is increasingly affected by various anthropogenic stressors that have resulted in continuous loss of biodiversity, such as the probable extinction of Yangtze River Dolphin. The Chinese paddlefish, Psephurus gladius, was one of only two extant members of a relict lineage that was most diverse and widespread 34–75 million years ago. It is also one of the largest freshwater fish species, reaching up to 7 m in length. The Chinese paddlefish was once common in the Yangtze River, with c.25 t being harvested per annum during the 1970s. Populations have, however, declined drastically since the late 1970s as a result of overfishing and habitat fragmentation. Here, a basin-wide capture survey during 2017–2018 found 332 fish species, but did not find a single specimen of Chinese paddlefish. Furthermore, 140 historically reported fish species have not been found and most of them are considered highly endangered. Based on 210 sightings of Chinese paddlefish during the period 1981–2003, we estimated the timing of extinction to be by 2005, and no later than by 2010. In addition, the paddlefish probably became functionally extinct (i.e. it was unable to reproduce) by 1993, before it went extinct. It is likely that the lack of reproduction was among the major causes of extinction. As no individuals exist in captivity, and no living tissues are conserved for potential resurrection, the fish should be considered extinct according to the IUCN Red List criteria. The delayed extinction of Chinese paddlefish resulted from multiple threats, suggesting that optimizing conservation efforts on endangered Yangtze fauna is urgently needed.