In vitro production of oocytes capable of producing offspring has exciting potential applications in reproductive medicine. Here, we generated and characterized an ovarian organoid model derived from female germline stem cells using a three-dimensional culture system. We show that this model generated normal offspring and detected drug toxicity. The ovarian organoids could produce oocytes and exhibited endocrine functions. Single-cell analysis of ovarian organoids identified six ovarian cell lineages, such as germ, granulosa and theca cells, and produced gene-expression signatures for each cell type. Investigation of the expression patterns of genes related to meiosis and gene ontogeny analysis for germ cell clusters showed that a germ cell population was maintained in the ovarian organoids. Moreover, flow cytometric analysis confirmed that the population of germ cells could be maintained on the organoids and showed that ascorbic acid treatment had a beneficial effect of germ cell population maintenance on the organoids. Furthermore, we demonstrated the successful production of offspring from oocytes derived from ovarian organoids. Finally, we showed the ovarian organoids had the potential to drug toxicological detection. For example, we found that salinomycin impaired the formation of ovarian organoids and germ cell population maintenance by inducing apoptosis. These results indicate that the female germline stem cell-derived ovarian organoids represent a valuable model system for generating oocytes that can yield offspring, and provide a novel model for drug screening and toxicological detection. Keywords: 3D culture; Female germline stem cell; Offspring production; Ovarian organoid; Single cell RNA-Seq; Toxicological detection.