Objective Mulberry brown spot disease and mulberry ring leaf spot disease are two common fungal diseases of mulberry trees (Morus alba), which are serious and cause great losses in production. This study was aimed to quickly and accurately diagnose the main pathogens of the two diseases, Neophloeospora maculans and Gonatophragmium triuniae.Method Based on the principle of multiplex PCR, specific primers for multiplex PCR were designed for the ribosome internal transcribed spacer (ITS) sequences of two pathogenic fungi, and the conditions of multiplex PCR reaction were optimized. Tests were performed with 45 samples to verify the feasibility of the established multiplex PCR.Result The established multiplex PCR detection system had good operability and good specificity, and the sensitivities for simultaneously detecting DNA of two pathogens were 0.1 and 1.0 pg/μL, respectively. The detection of mulberry disease samples could clearly distinguish the two types of pathogenic fungi in different regions. Conclusion The established multiplex PCR technology can be used for rapid detection of the pathogens of mulberry brown spot and ring leaf spot diseases, and provides a foundation for the prevention and control of mulberry fungal disease.