Objective To investigate the value of serum CA125 and human epididymis protein 4 (HE4) levels in the differential diagnosis of ovarian benign tumor and ovarian cancer. Methods CA125 and HE4 were quantitatively detected with microparticles enzyme immunoassay(MEIA) and ELISA in the sera from 56 healthy controls, 92 ovarian cancer patients, and 39 ovarian benign tumor patients respectively. Results The serum levels of both HE4 and CA125 were significantly higher (P <0.01) in ovarian cancer than those in benign tumor group and healthy control. There was no significant difference (P >0.05) between ovarian benign disease group and healthy control. Compared with that in benign ovarian disease group, the diagnostic sensitivity of serum HE level in ovarian cancer was 57.6%, lower than that (77.2%) of serum CA125; however, the diagnostic specificity (97%) of HE4 was higher than that (85.0%) of CA125. When combined detection of HE4 and CA125, either one or both higher than the reference value shall be deemed positive, ovarian cancer diagnostic sensitivity and specificity reached to 94.6% and 85.0% respectively. Conclusions The combined detection of serum CA125 and HE4 is useful in the diagnosis and differential diagnosis of ovarian cancer.