This article reports a case of a 59-year-old female patient presenting with hematochezia and lower abdominal pain. Colonoscopy revealed rectal ulcers, and histopathology showed acute inflammation, leading to an initial diagnosis of ulcerative proctitis. After one month of standard anti-inflammatory therapy without clinical improvement, the diagnosis was revised to amoebic proctitis through stool microscopy combined with targeted next-generation sequencing (tNGS). The patient showed rapid symptomatic relief following anti-amebic treatment. This case highlights the diagnostic challenges of Entamoeba histolytica proctitis, including nonspecific clinical manifestations, endoscopic and pathological findings lacking specificity, and the limited sensitivity of conventional pathogen detection methods. For patients with treatment-refractory intestinal ulcers, molecular diagnostic techniques should be actively employed to improve diagnostic accuracy and prevent misdiagnosis and inappropriate treatment.