The interaction of nanomaterials with biological macromolecules is an important foundation of the design and the biological safety assessments of nanomaterials. This work aims to investigate the interaction between pristine C60 and serum albumins (human serum albumin and bovine serum albumin) in solution. Stable aqueous dispersion of C60 was prepared by simple direct ultrasonic method and characterized by UV-vis spectrophotometry, transmission electronic microscopy and dynamic light scattering techniques, and spectroscopic methods (fluorescence spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy) were utilized for the investigation. It was found that the fluorescence of serum albumins could be quenched by C60 nanoparticles in a substantially similar way. Slight changes of the surrounding microenvironment of amino residues were observed, while little effects on the protein secondary structure occurred. The different effects of dispersion methods on the interaction of C60 nanoparticles with serum protein were also compared and discussed.