Ovarian cancer is a common gynecological malignant disease, causing more deaths among women .The key objective in the treatment of ovarian cancer is early diagnosis. The objective of our study was to seek new ovarian cancer biomarkers based on a serum protein profile with the aim of discriminating ovarian cancer patients from healthy controls. An MB-WCX kit was used to analyze serum samples obtained from 20 ovarian cancer patients and 20 healthy controls and then we generated MALDI-TOF protein profiles from the analysis. After pre-processing of the spectra, linear analysis with ClinProTools bioinformatics software was used to classify protein profiles and search for prominent peaks that could be used as potential ovarian cancer biomarkers. Using ClinproTools bioinformatics and statistical software, we found 5 prominent expressed proteins in the ovarian cancer and healthy control groups. The mass to charge ratio were 4648.21(m/z), 9294.03(m/z), 3886.1(m/z), 9066.38(m/z) and 4254.71(m/z), respectively, and the former four proteins were expressed higher in the ovarian cancer patients, but the later one was expressed at lower levels in the cancer patients. The sensitivity and specificity were both more than 90%. From our study, we found that MALDI-TOF MS is a high-throughput sample preparation method and is a new potential tool for the diagnosis of human disease, not only to search for new early detection biomarkers in the ovarian cancer patients' serum samples, but also with a potential use for routine clinical work.