The different range characteristics of acridine orange in absorption and fluorescence is dependent on its concentrations.The importance and mechanism as well as principles of choosing correct acridine orange concentration and rational amounts of lysosome in the assay of protons pumping are discussed. The obvious influences of the incubation time of acridine orange with lysosome and the K^＋/H^＋ exchange upon the measurements of proton transport are studied.