In this article, the interaction between bovine serum albumin (BSA) and the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) at the air-buffer interface was investigated at different subphase's pH values (pH = 3, 5 and 10). Surface pressure measurements (π - A) and penetration kinetics process (π - t) were carried out to reveal the interaction mechanism and the dynamical behavior. The data showed that π - A isotherms moved towards larger mean molecular area when the concentration of BSA ([BSA]) increased, the amount of BSA adsorbed onto DOTAP monolayer reached a threshold value at a [BSA] of 5 × 10-8 M, and BSA desorbed from the lipid monolayer as time goes by. The results revealed that the association of BSA with DOTAP at the air-buffer interface was affected by the subphase's pH value. When pH = 10, the interaction mechanism between them was a combination of hydrophobic interaction and electrostatic attraction, so BSA molecules could be well separated and purified from complex mixtures. AFM images demonstrated that pH value and [BSA] could affect the morphology feature of DOTAP monolayer and the adsorption and desorption processes of BSA. So the study provides an important experimental basis and theoretical support for learning the interaction mechanism among biomolecules in separation and purification of biomolecules and biosensor.