Numerous studies have shown that microRNAs (miRNAs) are important regulators of ovarian development in mammals but by comparison there is limited information on the role of miRNAs in ovarian physiology of teleost fishes. This study set out to examine dynamically expressed miRNAs in full grown (FG) zebrafish follicles when treated with the steroid hormone 17α, 20β-dihydroxy-4-pregnen-3-one (17α,20βP) which promotes oocyte maturation and ovulation. Using microarray analysis, 256 known zebrafish and the greatest changing mammalian miRNAs were identified in 17α, 20βP treated FG follicles with 18 upregulated and 6 downregulated. In vitro and in vivo experiments coupled with RT-qPCR demonstrated dre-miR-181a-5p, dre-miR-22a-3p and cgr-miR-1973 were differentially expressed in vitro while dre-miR-181a-5p and dre-miR-181c were differentially expressed in vivo between control and 17α, 20βP treated follicles. This study provides evidence for the presence and hormonal regulation of miRNAs in the zebrafish ovary, but further investigation is required to understand their role.