As post-transcriptional regulators, microRNAs (miRNAs) play an important role in growth and reproductive processes. So far, there is limited information regarding crustacean miRNAs. To explore the potential role of miRNAs in the gonadal development of the prawn Macrobrachium rosenbergii, we constructed seven small RNA libraries from ovarian and testicular tissues at various stages using somatic tissue as the control. A total of 1,954 known and 129 novel miRNAs were retrieved. By comparing differentially expressed miRNAs (DEMs) between testes and ovaries, forty-one miRNAs were identified with sex-biased expression patterns, including 17 ovary-biased and 24 testis-biased patterns. Furthermore, the putative target genes of the sex-biased miRNAs, such as cyclin L1, mitogen-activated protein kinase 7 (MAPK 7), heat shock protein (HSP), and zinc finger protein, were significantly enriched in many reproduction-related pathways including the Gonadotropin-releasing hormone (GnRH) pathway, glycolysis, gluconeogenesis pathway, ovarian steroidogenesis, estrogen signaling pathway, MAPK pathway, Wnt pathway, and insulin signaling pathway, implicating potential regulatory roles of miRNAs in reproduction. These data aid in the further investigation of the mechanism of gonadal development and reproductive regulation mediated by miRNA in M. rosenbergii.