The purpose of this study was to identify novel autoantibodies against tumor-associated antigens (TAAs) and explore a diagnostic panel for Ovarian cancer (OC). Enzyme-linked immunosorbent assay was used to detect the expression of five anti-TAA autoantibodies in the discovery (70 OC and 70 normal controls) and validation cohorts (128 OC and 128 normal controls). Machine learning methods were used to construct a diagnostic panel. Serum samples from 81 patients with benign ovarian disease were used to identify the specificity of anti-TAA autoantibodies for OC. In both the discovery and validation cohorts, the expression of anti-CFL1, anti-EZR, anti-CYPA, and anti-PFN1 was higher in patients with OC than that in normal controls. The area under the receiver operating characteristic curve, sensitivity, and specificity of the panel containing anti-CFL1, anti-EZR, and anti-CYPA were 0.762, 55.56%, and 81.31%. The panel identified 53.06%, 53.33%, and 51.11% of CA125 negative, HE4 negative and the Risk of Ovarian Malignancy Algorithm negative OC patients, respectively. The combination of the three anti-TAA autoantibodies can serve as a favorable diagnostic tool for OC and has the potential to be a complementary biomarker for CA125 and HE4 in the diagnosis of ovarian cancer.