Olive flounder (Paralichthys olivaceus) is an economically important cultured marine fish in China, Korea, and Japan. Controlling and managing the breeding of olive flounder in captivity is an imperative step toward obtaining a sustainable supply of this fish in aquaculture production systems. Therefore, investigation on the molecular regulatory mechanism of gonadal development and gametogenesis in this species is of great significance in aquaculture. Furthermore, identification of the expression profile of numerous sex-related genes is the first step to primarily understand such molecular regulatory mechanism. Six female and six male gonads obtained from 2-year-old olive flounders were sequenced using Illumina, which produced 6.68 and 6.24 GB data for ovary and testis, respectively. The reads were mapped to the draft genome of olive flounder, and then the reads per kilobase per million (FPKM) for each gene were calculated. The female-/male-biased expressed genes were investigated based on the FPKM values. Overall, 3086 female-biased and 5048 male-biased genes were screened out. GO enrichment analysis showed that the GO terms "male meiosis," "gamete generation," "fertilization," "spermatogenesis," and "germ plasma" were enriched in male-biased genes. In addition, the GO terms "cell morphogenesis involved in differentiation," "embryonic morphogenesis," "plasma membrane," "steroid hormone receptor activity," and "aromatase activity" were enriched in female-biased genes. Moreover, 373,369 single nucleotide polymorphisms and 32,993 indels were identified in the transcriptome. This work is the largest collection of gonad transcriptome data for olive flounder and provides an extensive resource for future gonadal development and gametogenesis molecular biology studies in this species.