Esophageal squamous cell carcinoma is a malignant tumor with unfavorable prognosis. In this study, we investigated the usefulness of microRNA (miR)-1246 detection in various body fluids as a biomarker for this disease. A total of 72 patients with esophageal squamous cell carcinoma were enrolled, and their blood, urine, and saliva samples were collected prior to treatment. Reverse transcription-polymerase chain reaction of miR-1246 was performed, and pre- and postoperative and intraday fluctuations in its expression were examined. The expression of miR-1246 in the blood and urine was significantly higher in the patients with esophageal squamous cell carcinoma than in 50 healthy control subjects. Receiver operating characteristic curves showed that the area under the curve values were 0.91 (sensitivity 91.7%, specificity 76.0%), 0.82 (sensitivity 90.3%, specificity 62.0%), and 0.80 (sensitivity 83.3%, specificity 66.0%) in the serum, urine, and saliva, respectively. A relatively high diagnostic performance of miR-1246 was observed in all samples, which was better than that of the existing biomarkers squamous cell carcinoma antigen, carcinoembryonic antigen, and cytokeratin 19 fragment. No clear correlation was observed in the levels of miR-1246 expression among the three body fluids. Postoperatively, serum samples displayed significantly decreased miR-1246 levels. Although not significant, changes in the miR-1246 levels were observed at all collection times, with large fluctuations in the saliva. Meanwhile, serum miR-1246 expression was found to be associated with the disease prognosis. The results indicate that the levels of miR-1246 in the urine, saliva, and serum are a useful biomarker for esophageal squamous cell carcinoma and support the use of urine samples instead of blood samples for noninvasive diagnosis.