Abstract: In Epinephelus coioides, foxl3 showed the expression characteristics of gender dimorphism, and the expression level of foxl3 in testis of E. coioides was significantly higher than that in ovary and other tissues. In addition, foxl3 is considered to play an important role in ovarian and testis development. In this study, by comparing the over-expression of foxl3 gene in E. coioides with 17ɑ-methyltestosterone (MT) feeding, explored a new method of regulating gonad development in E. coioides. The gonadal development status was observed and the genes related to gonadal differentiation were detected and analyzed by real-time PCR after the injection of foxl3 recombinant plasmid and MT feeding to three-month-old E. coioides. The results showed that the ovary of the injection group is smaller than that of the control group, and there are more somatic cells in the early stage of plasmid injection, and there is a trend of development in the male direction ( there were testicular interstitial cells ). The relative expression levels of male-related gene–amh, hormone-producing genes–cyp11b, 11β-hsd2, and spermatogenesis-related genes–rec8, sycp3 genes in the early injection group after plasmid injection are significantly higher than those in the control group. In particular, gene expression in amh, cyp11b and sycp3 injection groups was twice as high as that in control group. The gonads in the late MT feeding stage are in the obvious facultative stage, and the control group is still in the O2 oocyte stage. The relative expression trend of amh, cyp11b, 11β-hsd2, rec8 and sycp3 genes in MT feeding group is consistent with plasmid injection, but the difference between the feeding group and the control group is more significant than that of plasmid injection. Under the experimental conditions, the effect of MT feeding on the regulation of gonad masculinization is more obvious than that of foxl3 plasmid injection. In conclusion, the above results showed that the over-expression of foxl3 recombinant plasmid has a clear male regulatory effect on gonadal development of juvenile E. coioides, and this regulation may have a dose-dependent effect. The results laid the foundation for further study on testis development of E. coioides.