Abstract: Objective To evaluate serum human epididymal protein 4(HE4), carbohydrate antigen 125(CA125) and methylation of SEPT9(mSEPT9) in the urine in the diagnosis of endometrial cancer.Methods A total of 40 patients who were admitted to the Affiliated Hospital of Xuzhou Medical University and diagnosed with endometrial cancer were selected as a research group. Meanwhile, 39 patients who were diagnosed with benign endometrial lesions were selected as a control group. Their levels of serum HE4 and CA125 were detected by chemiluminescence. Their Ct values of mSEPT9 in the urine were detected by real-time fluorescent quantitative PCR.Results The research group showed remarkably higher levels of serum CA125 and HE4 than the control group(P<0.05). According to ROC curves, the cut-off values of serum CA125, HE4 and urine mSEPT9 Ct values were 14.65 kU/L, 52.44 pmol/L and 38.95, respectively. The research group presented significantly higher detection rates of serum CA125 and HE4 and urine mSEPT9 than the control group(P<0.05). According to ROC curves, the area under the curve of mSEPT9, CA125 and HE4 were all >0.5, with clinical diagnostic significance. The specificity of mSEPT9 was higher than those of HE4 and CA125. Furthermore, CA125 level was correlated with pathological stage, pathological grade and lymphatic vascular metastasis(P<0.05); HE4 level was correlated with age and depth of muscle infiltration(P<0.05); and the positive rate of mSEPT9 was correlated with pathological stage, depth of muscle infiltration, lymphatic and vascular metastasis(P<0.05).Conclusions Detection of urine mSEPT9 has great potential in the screening of endometrial cancer, prociding new thoughts for endometrial cancer screening.