Differentiating malignant from benign ascites often leads to confusion and an inability to exclude its multitude of causes in many patients. In the present study, T lymphocyte subsets and DNA ploidy in ascitic fluid were detected by flow cytometer. There were significant differences in T lymphocyte subsets between benign and malignant ascites. For malignant ascites, the sensitivity of DNA aneuploid is 75.6%, the specificity is 79.0%, and the accuracy is 77.6%. The results demonstrate that T lymphocyte subsets determination and DNA ploidy analysis can be used in the differential diagnosis between benign and malignant ascites.