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The interaction between gold nanoparticles (GNPs) and bovine haemoglobin (BHb) was studied by ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) and fluorescence spectroscopic techniques. The UV-Vis absorption spectrum demonstrated that there was interaction between GNPs and BHb, but no direct interaction between GNPs and haem groups of BHb. The fluorescence data revealed that GNPs effectively quenched the intrinsic fluorescence of BHb via static quenching. The binding of GNPs to BHb occurred at a single site. The binding process was a spontaneous molecular interaction procedure, in which hydrophobic force and hydrogen bonds played a major role. The alternations of protein secondary structure in the presence of GNPs were also determined by CD spectroscopy. This work is helpful to understand the interaction mechanism of GNPs with haemoglobin, which can guide the applications of GNPs in biomedicine.