Pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on Enterocytozoon hepatopenaei (EHP). Qualitative and Quantitative analysis was achieved by using Real time Loop mediated isothermal amplification (real-time LAMP) method. Optimization of amplification was attempted with three different temperatures (64, 66 and 68 °C) and accomplished with 68 °C. Sensitivity analysis was achieved in comparison with nested and real-time polymerase chain reaction to real-time LAMP. It has been proved to be without any cross reactivity with other aquatic pathogens like white spot syndrome virus, Infectious myonecrosis virus, Vibrio panaeicida and microsporidium seriolae. Real-time LAMP can be used in sensitive, specific and early detection of EHP in shrimp. This study was recorded as first attempt in detection and quantification of Enterocytozoon hepatopenaei in shrimp using real-time LAMP assay. © 2017 Elsevier B.V.