Ovarian cancer contains a microenvironment that supports the proliferation of cancer cells. The microenvironment of ovarian cancer can be produced in vitro by the manufacture of an ovarian cancer scaffold. The scaffold provides conditions structurally similar to the in vivo microenvironment for communication between cells and cells with the extracellular matrix (ECM) thus providing in vitro platform for disease modeling or drug screening. The decellularization method is used to remove cellular components from the ECM with little damage to the structure. The manufacture of ovarian cancer scaffolds in this study are carried out by physical and chemical decellularization techniques with comparison between one, two or three cycles of freeze-thawing. The process of making an ovarian cancer scaffold is using NaOH 0.4N with one, two or three times of freeze-thawing (−80 °C and 37 °C) followed by agitation with an orbital shaker. Characterization of the scaffold was performed by measurement of DNA concentration, histology analysis of decellularization by hematoxylin eosin and scanning electron microscopy (SEM), ECM analysis by masson trichrome for collagen area fraction, collagen III immunohistochemistry, fibronectin immunohistochemistry and byglican immunohistochemistry, cytotoxicity assay of SKOV-3 cell line using flow cytometry 7-AAD. The average genetic material of DNA from the scaffold is lowest at 4.1 ng/µl from the three times freeze-thawed cycles. Hematoxylin-Eosin staining showed a minimum amount of absence of nuclei and presence of intact ECM structure in fibers form particularly in the three times freeze-thawed