Increasing manufacturing and use of nanoparticles in industrial and biomedical applications creates the necessity to understand the impact of the interaction of nanoparticles with biomacromolecules. In the present study, graphene oxide nanosheets (GONS) were synthesized using modified Hummer's method and further characterized employing X-ray diffraction (XRD), UV, FTIR, and Raman spectroscopy. After characterization, the interaction of GONS with human serum albumin (HSA) was investigated to delineate the binding mechanism employing different kinds of spectroscopic techniques. Intrinsic fluorescence spectroscopy revealed that complex formation is taking place between HSA and GONS. Fluorescence-based binding studies suggested that GONS binds to HSA with a significant binding affinity, and the interaction is governed by dynamic quenching. The evaluation of enthalpy change (ΔH) and entropy change (ΔS) suggested that the HSA-GONS complex formation is driven by hydrogen bonding and van der Waals interaction and hence complexation process is seemingly specific. Structural transition in the microenvironment of HSA was monitored using synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy, which showed that GONS binding to HSA influences the microenvironment around tyrosine and tryptophan residues. Secondary structural alterations in HSA upon binding to GONS were measured using circular dichroism (CD) spectroscopy. Additionally, molecular docking provided an insight into the critical residues involved in HSA-GONS interaction and further validated our in vitro observations affirming interaction between GONS and HSA. The significance of this study is attributable to the fact that HSA and GONS can be used as nanocarriers in drug delivery systems. © 2021 Elsevier B.V.