Enterospora epinepheli are a major pathogen for cultured groupers (Epinephelus spp.),located on the coast of Fujian, Guangdong, Shandong, and Hainan Provinces, as well as the Guangxi Zhuang autonomous region, and can cause large economic losses to the grouper farming industry. Early and specific diagnosis is essential for the treatment and management of this emerging pathogen. However, since it was reported in 2017, there are only two nucleic acid-based techniques assays including PCR and qPCR assays, based on the TaqMan probe assay for the detection of E. epinepheli. This study developed a method for quick and accurate detection of E. epinepheli by loop-mediated isothermal amplification (LAMP) with three primer pairs by targeting the small subunit ribosomal RNA gene (SSU rRNA) of E. epinepheli (LAMP for E. epinepheli, E.ep_LAMP). The sensitivity, specificity, repeatability, and clinical applications of the developed E.ep_LAMP assay were verified. The constructed recombinant plasmid standard pMD18_E.ep, diluted with 10-fold gradient into different concentrations from 2.68×109 to 2.68×101 copies/μL, were used as a template to generate the standard curve and used to evaluate the detection sensitivity for E.ep_LAMP. The results showed that the detection limit of E.ep_LAMP was as low as 2.68×102 copies per reaction. The standard curve, y=–1.719 5x+23.971 (R² =0.996 2), was obtained by plotting the threshold cycle values (y) against the common logarithmic copies (log10 [Copy number] as x) of pMD18_E.ep plasmids template ranging from 2.68×10² to 2.68×10⁹ copies/μL. To assess the repeatability of the E.ep_LAMP, the intra-assay (variance within runs) and inter-assay (variance between runs) were evaluated, the coefficient of variation (CV), which are equal to the percentage of the standard deviation (SD) to the mean of the Ct, of intra-assay and inter-assay values were ranged from 0.22% to 3.52%, indicating that this assay was highly repeatable and reproducible over a wide range of detection from 2.68×10² to 2.68×10⁹ copies of the pMD18_E.ep plasmid template. To test the detection specificity of E.ep_LAMP, different gDNA extracted from different pathogens and diseased grouper tissue was used as templates. The results showed that the developed assay was also highly specific to E. epinepheli and had no cross-reaction with Enterocytozoon hepatopenaei (EHP), Ameson portunus, and another 11 pathogens, including Vibrio campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi, V. rotiferianus, V. natriegens, Spiroplasma eriocheiris, P. damselae subsp. damselae, white spot syndrome virus, decapod iridescent virus 1, and infectious hypodermal and hematopoietic necrosis. In validation of the E.ep_LAMP on clinical samples, a total of 50 g DNA samples extracted from clinical grouper samples were applied to detect the quantitation of E. epinepheli copies by the E.ep_LAMP assay, and to detect by the conventional PCR assays described in a previous report, respectively. The results showed that 50% of the total samples were detected positive using E.ep_LAMP, and loads of E. epinepheli in these positive clinical samples varied within the range from 4.54×10² to 3.23×10⁶ copies per mg tissue. By contrast, 42% of the total samples were detected positive using the PCR assay. These results showed that the E.ep_LAMP exhibited more sensitivity in detecting E. epinepheli in clinical samples. The E.ep_LAMP can be carried out on a simple heating device for incubation. By replacing the fluorescent dyes EvaGreen® with GeneFinder^® in the tubes of LAMP products, green fluorescence can be observed clearly with the naked eye in the positive control reaction tubes and E. epinepheli-infected samples, whereas it was orange in the negative control. We are currently exploring the development of a diagnostic kit based on this developed E.ep_LAMP assay. Taken together, the E.ep_LAMP diagnostic protocol developed in the present study was rapid, specific, and sensitive, and can be used as a diagnostic tool for the detection of microsporidian disease occurring in grouper culture.