Abstract: Background Metschnikowia bicuspidate is one of the pathogens of milky disease in Eriocheir sinensis, which is a serious obstacle to the development of aquaculture industry. So far there is no effective drug treatment. Rapid, efficient and accurate diagnosis is the key to prevention and control. Molecular biology techniques, such as PCR, have enabled safe and rapid detection of M. bicuspidate compared to traditional pathogen isolation and culture techniques. But it still requires complex temperature-changing equipment that makes it difficult to carry out testing at the poolside. Objective A new method based on hyperbranching rolling circle amplification (HRCA) technology has been established for detecting Saccharomyces cerevisiae, providing a new approach for pool-side detection of yeast pathogens. Methods Herein, HRCA was established based on the sequence of the 26S rRNA (U44822) of M. bicuspidate, by designing a padlock probe (PLP) and primers with high specificity. The sensitivity, specificity, reproducibility and application effect of the method were also evaluated. Results The results showed that the optimal reaction system for HRCA was 1 μmol/L probe concentration, 20 min amplification time, 63 ℃ amplification temperature, and 2 U/mL Bst DNA polymerase concentration. The method has a detection range of 0.50－1000 fmol/L for standard samples, exhibits high interspecies specificity, enables single base mismatch recognition, and demonstrates good repeatability and stability. In actual sample testing, the method had a sensitivity of 4.13×10¹ pg/μL for detecting DNA from hepatopancreas of Chinese mitten crabs and a detection rate of 56.25% for randomly selected Chinese mitten crabs, consistent with the results obtained using the qPCR method. Conclusion In conclusion, the hyper-branched rolling circle amplification technique can efficiently, rapidly and sensitively detect the ‘milky disease’ pathogen M. bicuspidate under constant temperature, which is suitable for poolside detection. The study not only provides a new means for the detection of yeast pathogens, but also provides new technical support for the early diagnosis and prevention of the disease, which has great potential for application in the field of aquatic pathogen detection and pathogen diagnosis.