Yellowfin seabream (Acanthopagrus latus) is a commercially important fish in Asian coastal waters. Although natural sex reversal has been described in yellowfin seabream, the mechanisms underlying sexual differentiation and gonadal development in this species remain unclear. MicroRNAs (miRNAs) have been shown to play crucial roles in gametogenesis and gonadal development. Here, two libraries of small RNAs, constructed from the testes and ovaries of yellowfin seabream, were sequenced. Across both gonads, we identified 324 conserved miRNAs and 92 novel miRNAs: 67 ovary-biased miRNAs, including the miR-200 families, the miR-29 families, miR-21, and miR-725; and 88 testis-biased miRNAs, including the let-7 families, the miR-10 families, miR-7, miR-9, and miR-202-3p. GO (Gene Ontology) annotations and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses of putative target genes indicated that many target genes were significantly enriched in the steroid biosynthesis pathway and in the reproductive process. Our integrated miRNA-mRNA analysis demonstrated a putative negatively correlated expression pattern in yellowfin seabream gonads. This study profiled the expression patterns of sex-biased miRNAs in yellowfin seabream gonads, and provided important molecular resources that will help to clarify the miRNA-mediated post-transcriptional regulation of sexual differentiation and gonadal development in this species.