This study presents a method to fabricate polydimethylsiloxane (PDMS) molds with hierarchical 3D structures by using the centric diatom, Coscinodiscus sp., as templates. The diatom frustules were immobilized on Si wafer, followed by casting the diluted PDMS on the substrate. After curing, the PDMS was peeled off from the Si wafer with the diatom frustules. Then it was etched in a hydrofluoric acid solution to dissolve the diatom frustules, so as to achieve the target replica mold. The parameters of replica molding process were optimized, such as the ratio of elastomer and cross-linker ratio, loading of diluents, and the time for vacuum-pumping. A PDMS negative replica opposite to the diatom frustules with micro/nano hierarchical structures were successfully obtained and the minimum structural size was ~40 nm. The detailed structure of the diatom frustules was further revealed through charactering the morphology of the replica. Meanwhile, the as-prepared replica may be used as molds for soft lithography of hierarchical structures.