The potential risk of nanomaterials to the environment and human health has caused widespread concernin recent years. Because of their small size and high reactivity, nanoparticles are likely to interact with biological macromolecules in vivo when they enter into the organisms. Therefore their original physical and chemical properties will change. Then, they are easily recognized and phagocytized by cells, and further bring about harm to the tissues and organs. In the process of interaction, biomacromolecules are also affected by nanoparticles, which may cause damage to their structure and interfere with the normal performance of their specific functions. Therefore, the toxic effect of nanomaterials on biomacromolecules is an important theoretical basis for studying their biosafety. Structure and function changes of human serum albumin (HSA) when exposed to carbon quantum dots (CQDs) and the mechanism for their interaction were studied by a variety of spectroscopic methods. According to fluorescence spectra and UV-Vis absorption spectra, CQDs quenched the intrinsic fluorescence of HSA by forming non-fluorescence complexes. Calculation results of binding constant (KA) and the number of binding sites (n) showed that there was only one binding site for CQDs on HSA, and synchronous fluorescence spectrum and binding sites competition experiment revealed this binding site was close to the only tryptophan residuein HSA, which located in ⅡA subdomain of protein. Based on Frster resonance energy transfer (FRET) theory, binding distance r between CQDs and HSA was calculated to be 2.89 nm, which is less than 8 nm, indicating that there was a high possibility of fluorescence quenching caused by non-radiative energy transfer between CQDs and HSA. From the calculated thermodynamic parameters, it can be inferred that hydrogen bonds and van der Waal’s force played an important role in the interaction of HSA with CQDs. According to the results of resonance light scattering(RLS) spectra, three-dimensional fluorescence spectra and circular dichroism (CD) spectra, CQDs could change the secondary and tertiary structure of HSA, causing α-helix conformation content of protein increase, promoting HSA further curling and folding, making hydrophobicity of microenvironment around tryptophan residue increase. Structural changes further affected the aggregation state and some physiological functions of protein, resulting in the decrease of the aggregation degree and esterase-like activity of HSA and a slight improvement of its free radical scavenging ability. This work can provide a reference for the evaluation of biological and environmental safety of nanomaterials, and it also provides a relatively systematic set of spectral analysis methods for exploring the interaction between nanoparticles and proteins.