miRNAs are a class of small non-coding RNAs that regulate gene expression. They are involved in the control of many developmental processes, including fruit development. The increasing amount of information on miRNAs, on their expression, abundance, and conservation between various species, provides a new opportunity to study the role of miRNAs in non-model plant species. In this work, we used a combination of Northern blot and tissue print hybridization analysis to identify conserved miRNAs expressed during prickly pear cactus (Opuntia ficus indica) fruit development. Comparative profiling detected the expression of 34 miRNAs, which were clustered in three different groups that were associated with the different phases of fruit development. Variation in the level of miRNA expression was observed. Gradual expression increase of several miRNAs was observed during fruit development, including miR164. miR164 was selected for stem-loop RT-PCR and for a detailed spatial-temporal expression analysis. At early floral stages, miR164 was mainly localized in meristematic tissues, boundaries and fusion zones, while it was more homogenously expressed in fruit tissues. Our results provide the first evidence of miRNA expression in the prickly pear cactus and provide the basis for future research on miRNAs in Opuntia. Moreover, our analyses suggest that miR164 plays different roles during prickly pear cactus fruit development.