Religious wildlife release is prevalent worldwide, especially in Asia countries. It is one of the anthropogenic pathways to cause biological invasions. Religious fish release is common on the Qinghai-Tibetan Plateau, yet few studies have assessed the influences of religious fish release on local species. In Yushu, a city on the Qinghai-Tibetan Plateau, we interviewed local people, conducted fish trap surveys in local rivers, and examined the diet of Eurasian otters Lutra lutra using a fecal DNA metabarcoding approach. We found that fish release started at least in 1980s -1990s in Yushu. Tibetan residents released fish in large amounts and released fish were usually exotic commercial fish purchased from market. Despite such long-term and intensive fish release activities, released fish were few in local rivers. On the other hand, Eurasian otters mainly prey on fish and released fish accounted for ~20% of relative read abundance of prey DNA in otters’ diet, indicating their high preference on released fish. Our study suggested that religious fish release may provide additional food resources for otters, whereas otters, as a top predator in local rivers, may deplete non-native fish once they were released and therefore reduce the probability of colonization of released fish, although further studies are required to assess otters’ impact. Our study revealed otters’ diet in Yushu, providing basic information for local otter management and conservation. Furthermore, it represents a case showing that native predators prey on religious released animals, implying a probable direction for controlling invasive species through native predator conservation.