The clinical treatment of ovarian cancer with ascites is problematic. The main reasons for treatment failure are the susceptibility to intraperitoneal metastasis and chemotherapeutic drug resistance. The purpose and significance of this study is to evaluate which marker might evaluate treatment efficacy and improve the cure rate. This study compared a no chemotherapy group with a chemotherapy group regarding the determination of carbohydrate antigen 125 and human epididymis protein 4 in ovarian cancer ascitic supernatants and cross-analyzed routine serum carbohydrate antigen 125 levels. The level of human epididymis protein 4 in the ascites of the chemotherapy group was significantly lower than that of the no chemotherapy group (p < 0.001). Moreover, the expression of ascitic human epididymis protein 4 correlated positively with serum carbohydrate antigen 125 levels (p < 0.001). MDR was positive in 13 of the 30 samples (43.33%) in the chemotherapy group with highly expressed CA125. The level of human epididymis protein 4 in ovarian cancer ascites may reflect the therapeutic effect of ovarian cancer patients, and a high level of human epididymis protein 4 might predict chemoresistance and the possibility of ascites formation. The determination of the expression of human epididymis protein 4 alone or combined with carbohydrate antigen 125 levels in both serum and ascites in ovarian cancer patients with ascites may have important significance for guiding and improving the treatment regimen.