Purpose This study aimed to evaluate trefoil factor 3 (TFF3), secreted frizzled-related protein 4 (sFRP4), reactive oxygen species modulator 1 (Romo1) and nuclear factor kappa B (NF-kappa B) as diagnostic and prognostic markers of endometrial cancer (EC) and ovarian cancer (OC). Methods Thirty-one patients with EC and 30 patients with OC undergone surgical treatment were enrolled together with 30 healthy controls in a prospective study. Commercial ELISA kits determined serum TFF-3, Romo-1, NF-kB and sFRP-4 concentrations. Results Serum TFF-3, Romo-1 and NF-kB levels were significantly higher in patients with EC and OC than those without cancer. Regarding EC, none of the serum biomarkers differs significantly between endometrial and non-endometrioid endometrial carcinomas. Mean serum TFF-3 and NF-kB levels were significantly higher in advanced stages. Increased serum levels of TFF-3 and NF-kB were found in those with a higher grade of the disease. Regarding OC, none of the serum biomarkers differed significantly among histological subtypes. Significantly increased serum levels of NF-kB were observed in patients with advanced-stage OC than those with stage I and II diseases. No difference in serum biomarker levels was found between those who had a recurrence and those who had not. The sensibility and specificity of these four biomarkers in discriminating EC and OC from the control group showed encouraging values, although no one reached 70%. Conclusions TFF-3, Romo-1, NF-kB and SFRP4 could represent new diagnostic and prognostic markers for OC and EC. Further studies are needed to validate our results.