The zebra-snout seahorse, Hippocampus barbouri, is an economically important marine fish and a potential candidate for aquaculture in Thailand. However, the reproductive ultrastructure of this seahorse is still poorly known. Transmission Electron Microscope (TEM) was used to characterize cellular morphology and ultrastructure of gametogenic stages in both sexes of H. barbouri. Based on morphology of the nucleus and unique characteristics of cytoplasmic organelles, oocytes in the oogenesis process of H. barbouri was classified into three distinct phases: primary growth phase (PG), secondary growth phase (SG) and atretic oocyte phase (AO). The early PG oocytes contained multiple nucleoli in close proximity to the nuclear membrane, showing the formation of cortical alveoli. The cytoplasm of late PG oocytes contained two distinctive cellular structures including lipid droplets and cortical alveoli and was enriched in rough endoplasmic reticulum, ribosome and mitochondria. The follicular complex complelety covered the oocyte at this phase and was classified into four distinct layers including zona pellucida, granulosa cells, basement membrane and theca cells. The yolk-granule formation was firstly observed in the early SG oocytes with well-developed microvilli in the zona pellucida and granulosa cells. During the late SG, the single-layered zona pellucida and the granulosa cells were well-organized. The dilated smooth endoplasmic reticulum and mitochondria were clearly visible in the granulosa cells. The AO oocytes exhibited disorganization of follicular complex. In male H. barbouri, spermatogonia and Sertoli-like cells occupied periphery of the germinal epithelium; however, the spermatocytes and spermatozoa were not observed in the germinal epithelium, possibly due to unique testis characteristics at this stage, season of the samples or aquaculture conditions. Taken together, this study unraveled characteristics of sexual differentiation in the zebra-snout seahorse and would provide benefit to monitor reproductive success of seahorse under aquaculture.