Soybean vacuolar H⁺-ATPase is one of the ATPases and play an important role in the growing period of the plant. Hypocrellin B and KI quench the intrinsic fluorescence of outside and inside membrane domain respectively. This two quench probes have been used to quench the protein’s intrinsic fluorescence under different pH and temperature. The relationship between hydrolysis activity and folding condition of V-ATPase has been preliminarily studied. The K_SV of outside and inside membrane domain under different pH and temperature had been compared. It shows that the intrinsic fluorescence of the protein and K_SV of outside and inside membrane domain all dropped with the deviation of pH and temperature from the optimum condition and the activity of the enzyme dropped too. This illustrates that the folding condition had been changed with the dropping of the enzyme’s activity. The changing of the folding condition of the protein plays an important role in the inactivation mechanism.