The fish-borne clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is endemic in a number of countries with over 35 million people being infected globally. Rapid and accurate detection of C. sinensis in its intermediate host fish is important for the control and prevention of clonorchiasis in areas where the disease is endemic. In the present study, we established a loop-mediated isothermal amplification (LAMP) approach for the sensitive and rapid detection of C. sinensis metacercariae in fish. The specificity and sensitivity of primers designed from the C. sinensis cathepsins B3 gene were evaluated, and specific amplification products were obtained with C. sinensis, while no amplification products were detected with DNA of related trematodes, demonstrating the specificity of the assay. The LAMP assay was proved to be 100 times more sensitive than a conventional polymerase chain reaction for detection of C. sinensis. The established LAMP assay provides a useful tool for the rapid and sensitive detection of C. sinensis in fish, which has important implications for the effective control of human clonorchiasis.