Objective: Malignant ascites (MA) of epithelial ovarian cancer (EOC) represents tumor microenvironment including immune system. The objective of this study was to analyze the tumor immune microenvironment using tumor-associated lymphocyte (TAL) isolated from MA in EOC and to observe changes after EOC relapse. Methods: We collected MA, peripheral blood and tumor samples a total of 95 patients (51 treatment naïve and 44 recurrent EOC). TALs from MA, peripheral blood mononuclear cells (PBMC) from the blood, and tumor-infiltrating lymphocyte (TIL) from the tumor were obtained. The immune phenotypes of TALs were compared with TILs using flow cytometry. Results: The percentages of CD8 T cells expressing PD-1, TIM-3, Ki-67, TOX, and CD101 in TAL were higher than those in PBMCs and similar to those in TILs. When we compared frequencies of terminally exhausted CXCR5-CD101 PD-1 CD8 T cells (Ttex) among PBMCs, TALs, and TILs of naïve patients, frequencies of Ttex were significantly higher in TILs than TALs and PBMCs and positively correlated with those in TILs. The exhaustion status of CD8 TALs was significantly correlated with that of CD8 TILs in terms of the expression of TCF-1 (r=0.40, p=0.047). Patients expressing high frequency of Ttex in TALs showed a trend towards poorer overall survival compared to those with lower frequency of Ttex. Conclusion: CD8 TALs demonstrated distinct immunologic properties compared to CD8 TILs. The frequencies of CXCR5-CD101 PD-1 CD8 Ttex in TAL could serve as a prognostic biomarker in patients with EOC. Further studies are needed to investigate the role of Ttex in MA of EOC.