Objective: To obtain the purified human ovarian normal fibroblasts (NFs) preliminarily. Methods: The ovarian tissues were dissociated by 0.25% trypsin, after overgrowing on the bottom of the culture dish,the cells were adhered to the bottom of the dish repeatedly to depurate them. Then the cells were verified according to its morphology under the inverted-microscope and were further confined by a series of biological markers with immunocytochemistry. And its growth cure was observed. Results: The typical shape of ovarian NFs was fusiform, cords like,triangular,polyangular and irregular. Most of them contain Pseudopods-like structure. The NFs showed positive staining for vimentin,fibroblast specific protein-1, desmin and α-smooth muscle actin,negative staining for cytokeratin pan,cytokeratin 7, cytokeratin 20, CD31, CD90, MMP-2, MMP-3, MMP-9, CA125, CA19-9, CEA, fibroblast activation proteinα (FAP-α) and platelet-derived growth factor receptorα(PDGFR-α). Conclusion: Human ovarian NFs were cultured successfully,which provides a tool for further studies on ovarian cancer and carcinoma associated fibroblasts.