Plenty of evidence showing that microRNAs (miRNAs) post-transcriptionally regulate gene expression and are involved in a wide range of biological processes. However, the roles of miRNAs in ovarian development process remain largely unknown in shrimp. In the present study, high-throughput sequencing of small RNAs was performed to find specific miRNAs that are involved in ovarian development process in Penaeus monodon. Two small RNA libraries were constructed from undeveloped (UNDEV group) and developed (DEV group) ovarian tissues in P. monodon. In total, 43 differentially expressed miRNAs were identified between the two groups (P ≤ 0.05, |log₂ ratio| ≥1), and their expression profiles were validated by qRT-PCR. In order to further clarify the functional roles of these differentially expressed miRNAs during ovarian development process, target gene prediction was performed. In total, 4,102 target genes of 43 miRNAs were predicted, then clustered by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database; only four specific pathways related to ovarian development were obtained (P < 0.05). Dual-luciferase reporter assays and integrated expression analysis were also conducted to further clarify the interaction between the miRNAs and their target mRNAs. This study provides important information about the function of miRNAs involved in ovarian developmental stages in P. monodon.