Abstract: The method for analyzing the interaction between caffeine and human serum albumin (HSA) was established by capillary electrophoresis. Under physiological conditions, the interaction between ligand (caffeine)-receptor (HSA) was studied with frontier-analysis (FA) method, Hummel-Dreyer (HD) method and plug-plug kinetic (PPK) method. The interaction parameters of caffeine-HSA system were obtained using non-linear equation, Scatchard equation and Klotz equation. The results showed that FA, HD and PPK methods were suitable for caffeine-HSA system, among them, HD method was the best, and the Non-linear equation was the best theoretical model to caffeine-HSA system. Interaction parameter tests showed that caffeine-HSA interaction was a single site interaction and the binding stability was moderate. The mechanism of caffeine-HSA interaction has been elucidated, which can provide valuable information for further research of alkaloids.