miRNAs are a class of small non-coding RNAs that regulate gene expression in eukaryotes. They are involved in the control of many plant developmental processes, including fruit development. The information available on miRNAs, related to expression, abundance, and conservation in several species, provides a new opportunity to study the role of miRNAs in non-model species, such as Opuntia ficus indica. In this work, bioinformatics and molecular biology methods were combined to isolate, identify and select candidate miRNAs in order to analyze their expression profiles during prickly pear development. The profiling comparison detected the expression of different miRNAs during prickly pear development, which were clustered in different expression patterns. A gradual increase in the expression of several miRNAs was observed during fruit development. The results described in this work, represent the first report of miRNA expression profiles during prickly pear fruit development; and provide the basis for future research on miRNAs on Opuntia.