MicroRNAs (miRNAs) play important roles in the regulation of genes associated with cancer development and progression. By the more deeply characterization of miRNAs' effect in cancer development, it requires a useful tool to investigate expression and distribution of a miRNA in cancer cells and tissues. To fulfill this application demand, we developed a miRNA in situ hybridization (MISH) approach using the 2'-Fluoro modified miRNA probe in combination with enzyme-labeled fluorescence (ELF) signal amplification approach. MISH was used to study expression of miR-375 in esophageal squamous cell carcinoma (ESCC) cell lines and tissues using a tissue microarray (TMA) containing 300 cases. The results showed that our MISH approach is a practical way to detect expression and distribution of a tested miRNA in both cultured cells and archive tissue sections. MISH results also showed that miR-375 was frequently downregulated in ESCCs, which was significantly associated with advanced clinical stage (p = 0.003) tumor metastasis (p = 0.04) and poor outcome (p = 0.04) of ESCC. Moreover, the accuracy of MISH results could be confirmed by QRT-PCR. Our results demonstrated that MISH is a useful and reliable tool to study miRNA expression in solid tumors. Downregulation of miR-375 can be used as a biomarker to predict the outcome of ESCC.