Thymoquinone (TQ) is the main constituent of Nigella sativa and is traditionally used as folk medicine. Our aim was to investigate the binding mechanism of TQ to human serum albumin (HSA) isoforms by utilizing biophysical methods such as intrinsic tryptophan fluorescence quenching, isothermal titration calorimetry (ITC), circular dichroism (CD), dynamic light scattering (DLS), Förster resonance energy transfer (FRET) and antioxidant activity in the absence and presence of TQ. We have calculated the binding and thermodynamic parameters from spectroscopic and calorimetric methods. When TQ interact with protein causes conformational change in secondary structure protein that was investigated by CD and change in radii of protein was monitor by DLS. Albumin itself have several types of activity, out of them esterase and antioxidant or radical scavenging activity were investigated with the both isoforms of HSA in the absence/presence of TQ. TQ has a very good antioxidant activity so that a remarkable enhancement in the antioxidant activity of HSA was obtained in the presence of TQ. Therefore, the efficiency of HSA to scavenge the free radical ions was increase in the presence of TQ which are generated in the body by various metabolic processes.