Background: Epithelial ovarian cancer (EOC) remains the most lethal disease among gynecological malignancies. Prompt diagnosis is challenging because of the non-specific symptoms exhibited during the early stage of the disease. So there is an urgent need for better detection methods. Here we performed this work to build up a platform of multiplex methylation-specific PCR (MSP) assay to improve the early detection of ovarian cancer, via identifying the methylation status of cell-free serum DNA. Methods: After screening, we chose seven genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5 and OPCML) with a high frequency of methylation as candidate genes to construct the multiplex-MSP assay. When methylation of at least one of the seven genes was observed, the multiplex-MSP assay was considered positive. We performed the retrospective and screening study to verify its specificity and sensitivity in the detection of EOC. Results: The methylation status of cell-free serum DNA was examined in the preoperative serum of 202 patients, including 87 EOC cases (stage I, n=41, stage II-IV, n=46), 53 benign ovarian tumors and 62 healthy controls. As expected, multiplex MSP assay achieved a sensitivity of 85.3% and a specificity of 90.5% in stage I EOC, strikingly higher than that of single CA125, producing a sensitivity of 56.1% at 64.15% specificity [p=0.0036](Table). Conclusions: Multiplex MSP assay analyzing the methylation status of cell-free serum DNA is a suitable and reliable approach to improve the early detection of ovarian cancer, potentially benefiting a broad range of applications in clinical oncology.