Regurgitated pellets (n = 584) of Long-eared Owl (Asio otus) were collected for three seasons from Gansu Endangered Animal Research Center, Gansu Province, China. From these pellets, a total of 1011 individual prey items – representing seven species of rodents and two undetermined species of birds and one undetermined sorex-species – were identified. Long-eared Owls depend mainly on small mammals both based on prey numbers (95.5%) and prey biomass (97.0%). The composition of the diet of Long-eared Owls varied significantly among seasons by frequency and by biomass. The individual species groups indicated significant variation among seasons for Mus musculus, Meriones meridianus, Cricetulus barabensis, Phodopus roborovskii, Dipus sagitta and Rattus norvegicus by biomass. And the difference of seasonal variation of prey items were significant but not for Mus musculus by frequency. Based on prey numbers, Mus musculus was the main food item (50.5%) in its diet; this species was most common in autumn (56.9%) and least common during spring (36.7%). Main prey species, based on prey biomass, was Rattus norvrgicus which comprised 27.5% of total biomass from our pellet samples. The mean length of tibias of mammals in pellets of Long-eared Owls was 18.6 ± 5.4 mm. Long-eared Owls utilize a wide range of prey items in respect to their habitats. Results suggest that Long-eared Owls are selective predators at some levels.