In the present paper, the interaction of bovine hemoglobin (BHb) and realgar nanoparticles has been investigated by ultraviolet-visible (UV-Vis) and fluorescence spectroscopy. The Soret band of oxygen-BHb at 406 nm shifted to 413 nm, and its absorption intensity decreased gradually after adding realgar nanoparticles. The Soret band decreases gradually with the increasing the amount of realgar NPs, suggesting the detachment of some heme chromophores from their matrixes in BHb. The red-shift of characteristic peak leads to be conjecture that the arsenic of realgar combined with the oxygen of BHb. The oxygen-BHb was deoxidated by realgar nanoparticles, and the surface binding induces conformation change of BHb from the high-affinity R state to the low-affinity T state. The fluorescence intensity of BHb is quenched by realgar nanoparticles when its concentration gradually increased. The analysis of Stern-Volmer equation revealed that the mechanism was a static quenching procedure. The order of the magnitude of binding constant k was 10(9), obtained from the calculation of UV-Vis and fluorescence spectra.