Ortleppascaris sinensis is the dominant nematode species infecting the gastrointestinal tract of the captive Chinese alligator, a critically endangered species. Gastrointestinal nematode infection may cause a loss of appetite, growth, a development disorder, and even mortality in alligators, especially young ones. This research first establishment a loop-mediated isothermal amplification (LAMP) assay in rapidly identifying O. sinensis, upon the basis of the complete internal transcribed spacers (ITS) gene. Eight sets of primers were designed for recognition of the unique conserved ITS gene sequences, and one set was selected to be the most suitable primer for rapid detection. The specific as well as the sensitive features of the most appropriate primer in LAMP reactions for O. sinensis, and feces specimens of Chinese alligators suffering from O. sinensis were determined. Turbidity monitoring and Te Visual Reagent methods were used for determining negative and positive consequences. According to this study, amplification and visualization of the target DNA could be realized through two detection approaches during 50 min at 65 °C isothermal temperature. The sensitivity of LAMP was a detecting limitation of 3.46 pg/µl DNA. No cross-reactions were found between O. sinensis and any other of the nine heterologous nematode parasites, which shows the outstanding specific features of the primers. The LAMP assay could also perform a detection of target DNA of O. sinensis in the feces samples of Chinese alligators. This LAMP assay is useful for directly detecting O. sinensis in the Chinese alligator breeding centers, particularly due to its rapidity, simplicity and low cost.