Fullerenols, known as polyhydroxylated derivatives of fullerene, have attracted great attention due to their distinctive material properties and potential applications in biology and medicine. As a step toward the elucidation of basic behavior in biological systems, a variety of spectroscopic measurements as well as isothermal titration calorimetry (ITC) were applied to study the interaction between fullerenol (C60(OH)44) and serum proteins (bovine serum albumin (BSA) and γ-globulins). The results of fluorescence spectra indicated that the intrinsic fluorescence of proteins could be effectively quenched by the dynamic mechanism. The affinity values of both proteins bound to fullerenol were of the same order of magnitude. Meanwhile, ITC results showed that the interaction between fullerenol and BSA was enthalpy favorable, while the interaction with γ-globulins was enthalpy unfavorable. Furthermore, fullerenol had little influence on the secondary structure of both proteins. Additional cytotoxicity tests showed that the presence of proteins attenuated the toxic effect of fullerenol on human normal gastric epithelial cell line (GES-1). Thus, the interaction between fullerenol and proteins is indispensable to evaluate the biosafety of fullerenol, which may in turn promotes the development of its biological applications.