Abstract: In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture, especially in fish with relatively long generations. Nevertheless, functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish. In this study, we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers (Bostrychus sinensis), which are prone to ovotesticular or sterile testicular development, and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional (3D) culture system. Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae. Furthermore, melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway, and thus increased the efficiency in functional sperm production. The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.