[Objective] To establish a detection system of soft-shelled turtle iridovirus (STIV, a causative agent of severe diseases in cultured Trionyx sinensis) with hyper-branched rolling cycle amplification (HRCA) assay, which provides a stable, reliable and novel technique for early diagnosis of the disease and identification of the pathogen. [Methods] The padlock probe consists of a universal linking sequence and the two target complementary regions at 5′ and 3′ ends, which was designed based on the unique fragment sequence of STIV, was synthesized. Detection system of HRCA was established and optimized. The specificity and sensitivity of HRCA was determined and compared with conventional PCR. Sick turtles with or without obvious symptom were detected applying HRCA. [Results] HRCA is capable of amplifying STIV DNA, while can not detect related virus and other aquatic animal virus. The detection sensitivity of HRCA is as low as 101 copy which is 100-fold higher than that of conventional PCR. All the detections of the infected soft-shell turtles with or yet without evident symptoms at early stage of infection showed positive results. [Conclusion] The detection system of HRCA for STIV was successfully established. It is sensitive, specific, rapid and simple. It can be used for early diagnosis with good prospects for generalizing in the turtle culture.