Retinal pigment epithelium (RPE) cells are exclusive to the retina, critically multifunctional in maintaining the visual functions and health of photoreceptors and the retina. Despite their vital functions throughout lifetime, RPE cells lack regenerative capacity, rendering them vulnerable and central to degenerative retinal diseases. With advancements in stem cell technology enabling the differentiation of functional cells from pluripotent stem cells and leveraging the robust autocrine and paracrine functions of RPE cells, extracellular vesicles (EVs) secreted by RPE cells hold significant therapeutic potential in supplementing RPE cell activity. While previous research has primarily focused on the trophic factors secreted by RPE cells, there is a lack of studies investigating miRNA, which serves as a master regulator of gene expression. Profiling and defining the functional role of miRNA contained within RPE-secreted EVs is critical as it constitutes a necessary step in identifying the optimal phenotype of the EV secreting cell and understanding biological cargo of EVs to develop EV-based therapeutics. In this study, we present a comprehensive profile of miRNA in small extracellular vesicles (sEV) secreted during RPE maturation following differentiation from human embryonic stem cells (hESCs). This exploration is essential for ongoing efforts to develop and optimize EV-based intraocular therapeutics utilizing RPE-secreted EVs, which may significantly impact the function of dysfunctional RPE cells.