The separation of primary amino acids in Radix Isatidis and its injection is performed by coordination interaction capillary electrophoresis with nonderivative direct UV detection. This study investigates the effect of concentration of Cu2+ and acidity of electrophoretic running buffer, separation voltage, injection time and the internal diameter of the capillary on separation and detection. Under the optimal operation condition, the two amino acids are qualitatively and quantitatively analyzed and its liner range, detection limit and reproducibility are investigated respectively. The results show that the content of arginine is 0.63 mmol/L and the content of proline is 25.7 mmol/L in the Radix Isatidis Injection. Under the same condition, the components of Radix Isatidis extracts are separated by the presented method. The mass fraction of arginine is 0.027 and the mass fraction of proline is 0.054.