Recently, post-transcriptional gene regulation by microRNAs (miRNAs) has been reported to play a key role during ovary development and differentiation. However, there are no published studies identifying miRNA profiles of human ovarian tissues directly using next-generation sequencing technology. In the human ovary, a total of 762 known and 21 novel human miRNAs were detected, indicating that human ovaries have a complex population of small RNAs. To confirm the miRNA profile in human ovaries, quantitative real-time polymerase chain reaction was used to validate the expression of known miRNAs and novel miRNAs. The potential regulating roles of miRNA in physiological function of ovaries were analyzed by gene ontology and Kyoto encyclopedia of genes and genomes pathway annotation, and several important processes were identified to be targeted by the most abundantly expressed miRNAs, for example, antral ovarian follicle growth, ovarian follicle rupture, and fertilization. Our current findings extend the knowledge of the regulatory role of miRNAs and their targeted processes in human ovaries, suggesting that miRNAs play important roles in development and physiological function of ovaries. In this study, we provide a useful resource for further research of the regulatory role of miRNAs in the ovaries, which may also provide novel candidates for molecular biomarkers or treatment targets in the research of female infertility.