The tryptophan fluorescence of a subunit of F_o moiety of mitochondrial F₁F_o can be quenched by the addition of Hypocrellin B. The determination of the Stern-Volmer plot at different temperatures is carried out. The result shows that the K_sv increased with the increase of temperature. The experimental result of the time-resolved fluorescence decay shows the decrease of lifetime of tryptophan fluorescence of F_o with the increase of concentration of HB. No shift in the absorbance spectra of F₁F_o were occurrent at varying concentration of HB. The results support the dynamic quenching. In addition, HB possess the necessary characters to be used as a quencher in the hydrophobic phase as follows: the low concentration of effective quenching; no effect on the activity of F₁F_o; the ratio of the partition coefficients between lipid-phase and water-phase as high as 16 560∶1. So HB can be used as a ideal fluorescence quencher for the study of conformational change of F_o moiety of membrane-bound F₁F_o complex.