MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and play key roles in many biological processes, such as development and response to multiple stresses. However, little is known about their roles in generating novel phenotypes and phenotypic variation during the course of animal evolution. Here, we, for the first time, characterized the miRNAs of the cochlea in an echolocating bat (Rhinolophus affinis). We sampled eight individuals from two R. affinis subspecies with significant echolocation call frequency differences. We identified 365 miRNAs and 121 of them were novel. By searching sequences of these miRNAs precursors in multiple high-quality mammal genomes, no specific miRNAs were found to be shared by all echolocating mammals. Together with the matched mRNA-seq data, we identified 1,766 differentially expressed genes (DEGs) between the two subspecies and 555 of them were negatively regulated by differentially expressed miRNAs (DEMs). We found that almost half of known hearing genes in the list of all DEGs were regulated negatively by DEMs, suggesting an important role of miRNAs in call frequency variation of the two subspecies. These targeted DEGs included several important hearing genes (e.g. Piezo1, Piezo2 and CDH23) that have been shown to be important in ultrasonic hearing of echolocating mammals.