With the advent of molybdenum disulfide nanosheets (MoS2 NSs) for biological applications, their complex interactions with human serum albumin (HSA) need to be understood in great detail for the molecular mechanisms of protein structure and activity. It was observed that MoS2 NSs quench the intrinsic fluorescence of HSA as a consequence of ground-state complex formation by the electron transfer, van der Waals, and hydrophobic forces. The presence of MoS2 NSs partly altered the conformation of HSA and destroyed the binding domain of HSA with bilirubin. In addition, MoS2 NSs can decrease the rate of the formation of beta sheet structures of HSA, reduce the non-enzymatic glycosylation, and increase the esterase-like activity of HSA. We hope that the present study will be helpful to understand the fundamental interactions of the two-dimensional materials with various biomacromolecules in human blood.