ABSTRACT: The function of microRNAs (miRs) is associated with the development and progression of various malignancies, with miRs presenting stably in the serum. The current study assessed the role of miR-1246 and miR-106b in the serum of patients with esophageal squamous cell carcinoma (ESCC). A comprehensive microarray analysis of miR expression was performed using the serum of patients with ESCC, which were subsequently validated via reverse transcription-quantitative PCR. A total of 55 test samples were obtained from Chiba University and 101 validation samples were gained from Chiba Cancer Center. The results revealed that miR-1246 expression significantly increased and miR-106b expression significantly decreased in each cohort. Receiver operating characteristic analysis revealed that the area under the curve (AUC) value of miR-1246 was 0.816 (sensitivity, 72.7%; specificity, 69.2%) and 0.779 (sensitivity, 71.3%; specificity, 70.6%) for the test and validation cohorts, respectively. The AUC of miR-106b was 0.716 (sensitivity, 65.5%; specificity, 61.6%) and 0.815 (sensitivity, 74.3%; specificity, 73.5%), respectively. In addition, the AUC of the miR-1246/miR-106b ratio was 0.901 (sensitivity, 80.0%; specificity, 80.0%) and 0.903 (sensitivity, 82.1%; specificity, 82.3%), respectively, which indicated a higher diagnostic ability compared with that of miR-1246 or miR-106b alone. The high miR-1246/miR-106b ratio group was associated with clinicopathological factors such as depth of invasion, progression, lymph node metastasis, and poor prognosis. Therefore, effective biomarkers may be generated by combining individual miRs obtained by comprehensive analysis of ESCC patient sera.