MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.