Enterocytozoon hepatopenaei (EHP) is a pathogen in the pancreatic tissue of prawn, as reported in recent years. Enterosporidiosis caused by EHP in Penaeus genus is spreading widely, which seriously threatens the sustainable development of shrimp aquaculture in the world. Empolying the DNA binding dye of SYTO-16, a real-time quantitative loop-mediated isothermal amplification (LAMP) method has been established herein to detect EHP. The primer sequences used in the LAMP reaction were according to the SSU rRNA gene. The LAMP assay has reached a sensitivity of 101 copies/µL and no cross-reaction with other aquatic pathogens. Compared with normal PCR, the efficiency of the LAMP technique is more sensitive, which has a shorter detection time. The use of fluorescent nucleic acid dye (SYTO-16) reveals a more satisfactory performance relative to calcein. The quantitative LAMP assay can be considered as a valid tool for rapid detection of microsporidian in prawns. Our study provides a scientific basis to follow the effect of the pathogen infection on growth of cultured penaeid shrimp.