Fluorescence quenching method was to measure the proton pumping activity of tonoplast H^＋-ATPase from higher plants. The sensitivity of two common fluorescent dyes was compared. Acridine orange is better than quinacrine with the initial slope and maximum quenching. The optimim for the fluorescence measurement of acridine orange dye were:5 μmol/L acridine orange, 200～250 μg protein, Hepes-Tris of Hepes-BTP(pH 7.0). Initiating with ATP or Mg^2＋ was both valid.