This study used different methods to establish an animal model of orthotopic transplantation for ovarian cancer to provide an accurate simulation of the mechanism by which tumor occurs and develops in the human body. We implanted 4T1 breast cancer cells stably-transfected with luciferase into BALB/c mice by using three types of orthotopic transplantation methodologies: (1) cultured cells were directly injected into the mouse ovary; (2) cell suspension was initially implanted under the skin of the mouse neck; after tumor mass formed, the tumor was removed and ground into cell suspension, which was then injected into the mouse ovary; and (3) a subcutaneous tumor mass was first generated, removed, and cut into small pieces, which were directly implanted into the mouse ovary. After these models were established, in vivo luminescence imaging was performed. Results and data were compared among groups. Orthotopic transplantation model established with subcutaneous tumor piece implantation showed a better simulation of tumor development and invasion in mice. This model also displayed negligible response to artificial factors. This study successfully established an orthotopic transplantation model of ovarian cancer with high rates of tumor formation and metastasis by using subcutaneous tumor pieces. This study also provided a methodological basis for future establishment of an animal model of ovarian cancer in humans.