Objective To establish a measurement method of peritoneal fluid volume in mice and evaluation of the water load in mouse models of kidney-yang deficiency syndrome in Chinese medicine.Methods To improve the measurement method of peritoneal fluid volume in mice based on Archimedes' principle. Twelve normal mice were selected to evaluate the measurement stability of peritoneal fluid volume. Five normal mice were selected for study of the linear relationship of ascites detection method. Thirty SPF male Kunming mice were randomly divided into control group and kidney-yang deficiency model group. The mouse models of kidney-yang deficiency were generated by intraperitoneal injection of estradiol benzoate in a dose of 0.08 mg/10 g in the morning for consecutive 15 days. The mice of normal control group were injected the same dose of soybean oil. All mice of the both groups received intraperitoneal injection of 1 mL saline in the afternoon to make water load. Results Good effect of the measurement stability and linear relationship study was achieved. Compared with the control group, the body weight of mice after modeling was significantly decreased, rectal temperature significantly declined (P<0.01), spontaneous motor activity was significantly reduced (P<0.05), and swimming time was significantly shorter (P<0.01). All the signs indicated that the kidney-yang deficiency was successfully produced in the mice. The peritoneal fluid volume in mice of the kidney-yang deficiency model group showed a decreasing trend, and the ascites index was significantly higher than that of the control group (P<0.05). Conclusions The established detection method can quickly and accurately measure the mice peritoneal cavity fluid volume and changes of the ascites index. It can be used to objectively evaluate the characteristics of TCM syndrome and provide experimental data for the TCM symptom description.