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Root rot is a highly infectious disease in traditional Chinese medicine (TCM) that spreads quickly and significantly affects the yield and quality of TCM. Therefore, there is an urgent need for accurate and convenient early warning technology to guide root rot control. The root rot of TCM is mainly caused by Fusarium fungi infecting plants. After plants have been infected with fungi, zearalenone (ZEN), fumonisin (FB), fusarium acid (FA), and other compounds will produce duo to Fusarium infects plants to promote metabolism. And the trace expression of these compounds in plants is used as root rot early warning markers (RREWM). Herein, an electrochemical immunosensor was developed for the rapid, convenient, and sensitive detection of zearalenone (ZEN), an early warning marker for Radix isatidis root rot. The immunosensor utilizes Fe3O4@g-C3N4 composite ITO electrode. Initially, the ITO conductive films were modified with Fe3O4@g-C3N4 and gold nanoparticles (AuNPs) through droplet coating and chemical deposition, respectively. Subsequently, the AuNPs were conjugated with zearalenone antibody (ZEN-Ab) to generate high affinity sulfhydryl-bound recognition elements. Under the optimized condition, the sensor demonstrates a linear wide range of 10.00 pg mL−1 to 1.20 × 104 pg mL−1 with a LOD of 3.86 pg mL−1. Additionally, the sensor showed good reproducibility and stability. This study exhibits a convenient, accurate, and low-cost method for detecting early warning markers of root rot in TCM cultivation, which was expected to provide a reliable warning technology for the early warning and prevention of diseases in the planting process of TCM. [ABSTRACT FROM AUTHOR]