Objective To optimize in situ hybridization (ISH) method for microRNA (miR) detection in formalin-fixed and paraffin-embedded (FFPE) pancreatic cancer tissue sections, so as to improve the sensitivity of miR detection. Methods Tissue microarray (TMA) was constructed from 20 pancreatic cancer tissue specimens. Using locked nucleic acid (LNA) labeled probe, we examined the expression levels of miR-21 and miR-34a by chromogenic in situ hybridization (CISH). The hybridization temperatures were set at 48, 53 and 56℃, respectively. Three different washing approaches (temperature, time and 3 different washing buffers) were adopted to optimize the hybridization conditions. TMA was prepared using 126 pancreatic cancer tissue specimens, and optimal CISH was used to detect the expression levels of miR-21 and miR-34a. Results The optimal hybridization conditions of miR-21 probe were as follows:hybridization at 53℃ for 6 h, washing with washing buffer Ⅲ at 65℃ for 6 min, and then washing with PBS for 1 min. The optimal hybridization conditions of miR-34a probe were as follows:hybridization at 53℃ for 6 h, washing with washing buffer Ⅲ at 4℃ and 65℃ for 6 min, respectively, and then washing with PBS for 1 min. Of 126 pancreatic cancer tissue specimens, 84 (66.7%) had positive expression of miR-21, and 77 (61.1%) had positive expression of miR-34a. Conclusion The optimal hybridization temperatures of miR-21 and miR-34a are both 53℃ in the FFPE pancreatic cancer tissue sections, and appropriate washing temperature and washing buffer after hybridization are conducive to improve the positive detection rate of miR.