Objective: This study aimed to investigate the predictive value of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for chemosensitivity test in ascites. Materials and Methods: The relationship of the in vitro sensitivity results and the clinicopathological characteristics, objective response rates (ORRs) of chemotherapy, and time to progression (TTP) were retrospectively analyzed in 120 epithelial ovarian cancer (EOC) patients. The clinical response criterion was based on the Response Evaluation Criteria in Solid Tumors (RECIST) standard. The log-rank test and Kaplan-Meier curve were used to estimate TTP. Results: MTT assays revealed that tumor cells from ascites of primary and type II EOC were more sensitive to paclitaxel (PTX) and carboplatin (CBDCA) than relapse (p = 0.01 and p < 0.01, respectively) and type I (p = 0.03, p = >0.02, respectively) EOC. p53 positive expression and Ki67 high expression were associated with high PTX (p = 0.01 and p < 0.01, respectively) and CBDCA (p = 0.03 and p < 0.01, respectively) sensitivity. Ki67 weak positive immunostaining was associated with topotecan (p < 0.01), gemcitabine (p < 0.01), and doxorubicin (p < 0.01) resistance. Chemosensitivity to CBDCA/PTX was associated with the ORR of neo-adjuvant (p = 0.03) and adjuvant (p = 0.02) chemotherapy. The MTT assay results of ascites were consistent with the clinical response (p = 0.04) and TTP (p = 0.04) in patients with platinum-resistant relapse EOC tumors. Conclusions: Evaluation of the chemosensitivity of ascites in EOC by MTT can aid the establishment of individualized clinical chemotherapeutic plans for platinum-resistant relapse patients.