Artificial induction and breeding of triploids has been widely performed in fish due to their sterility or low fecundity. However, detailed studies on the gonadal development status, gamete formation, fertilization ability, and molecular mechanism of the induced triploids in marine fish are limited. Olive flounder Paralichthys olivaceus is an important mariculture fish in China, Japan, and Korea. In this study, by observing the gonadal development of the artificially induced triploid and control diploid flounder, the histological structure, sex hormone level, and transcriptome profiles were compared. The fecundity of the male triploid fish was also evaluated. The results showed that the development of the gonads of the triploids was severely inhibited. Apoptosis occurred in both male and female triploid gonads, which may lead to inhibition of germ cell development. At 24 months post hatch (mph), the contents of testosterone (T) and estradiol (E2) in the gonads of the female triploid flounder were lower than those of the diploid ones (P < 0.05), and the contents of E2 in the testes of the male triploid flounder were higher than those of the diploid ones (P mns1, dnah1, and dnah5, were down-regulated; while in the triploid ovary, genes related to testicular development, such as amh, amhr2, and wnt4, were up-regulated, which also provided molecular evidence for the testicular-like gonads observed in the histology of the triploid ovary. These results may help explain the developmental retardation and low fecundity in the gonads of triploid fish that are important for the future application of culture.