Characterization of a newly established serous ovarian cancer cell line, YDOV-139 was performed and ER-60 (PDIA3), which was highly expressed in YDOV-139, was evaluated as novel biomarker for ovarian cancer. The YDOV-139 cell line was established using ascites samples from a 67-year-old Korean woman with recurrent ovarian cancer, and was characterized with respect to various biological and genetic features. Gene expression profiles were analyzed using cDNA microarrays, and proteomic evaluation was performed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting (MALDI-TOF/PMF). Four candidate markers that were strongly up-regulated in YDOV-139 were validated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). The epithelial-like characteristics of YDOV-139 were evident from morphologic studies, and the average population doubling time was 120 h. When transplanted into nude mice, YDOV-139 cells successfully induced tumor masses in all three animals. Chemosensitivity tests showed that gemcitabine had the highest chemosensitivity index against YDOV-139 cells. HLA typing revealed A*24/A*31, B*07/B*35, Cw03*(09)/w*07, and DRB1*01/DRB1*15 alleles. Compared with human ovarian surface epithelial (HOSE) cells, 2,520 genes and 23 protein spots were differentially expressed in YDOV-139. Validation by real-time PCR showed that mRNA expression of LCN2, MDK, SLCO4A1, and ER-60 (PDIA3) were strongly elevated in ovarian cancers. In IHC analysis, ER-60 (PDIA3) was significantly overexpressed in both borderline tumors and invasive ovarian cancers (P<0.001). The molecular characteristics of YDOV-139 may have implications for future ovarian cancer research and ER-60 (PDIA3) should be investigated further as a potential biomarker of ovarian cancer.