This study aimed to derive mature oocytes from murine preantral follicles cultured in a biomimetic ovary with a porcine scaffold using decellularization technology. We evaluated the DNA content and the presence of cell and extracellular matrix (ECM) components, including collagen, elastin, and glycosaminoglycans (GAGs), in decellularized (decell) porcine ovaries. The DNA content inthe decell ovarian tissues was approximately 94 % less than that in native tissues (66 ± 9.8 ng/mg vs. 1139 ± 269 ng/mg). Furthermore, the ECM component integrity was maintained in the decell ovarian tissue. The soluble collagen concentration of native ovarian tissue (native) was 195.34 ± 15.13 μg/mg (dry wt.), which was less than 878.6 ± 8.24 μg/mg for the decell ovarian tissue due to the loss of cellular mass. Hydrogels derived from decell porcine ovaries were prepared to develop an in vitro biomimetic ovary with appropriate ECM concentration (2−6 mg/mL). Scanning electron microscope (SEM) imagining revealed that the complex fiber network and porous structure were maintained in all groups treated with varying ECM concentration (2–6 mg/mL). Furthermore, rheometer analysis indicated that mechanical strength increased with ECM concentration in a dose-dependently. The preantral follicles cultured with 4 mg/mL ECM showed high rates of antral follicle (66 %) and mature oocyte (metaphase II) development (47 %). The preantral follicles cultured in a biomimetic ovary with a decell porcine scaffold showed a higher rate of antral follicle and mature oocytes than those cultured in other biomaterials such as collagen and Matrigel. In mature oocytes derived from antral follicles, meiotic spindles and nuclei were stained using a tubulin antibody and Hoechst, respectively. Two-cell embryos were developed from MII oocytes following parthenogenetic activation. Preantral follicles were cultured in a biomimetic ovary derived from the ECM of a decell porcine ovary, and embryos were generated from MII oocytes. This biomimetic ovary could contribute to restoring fertility in infertile women with reduced ovarian function, benefit mating efforts for endangered species, and maintain animals with valuable genetic traits.