Protease inhibitors (PIs) are a ubiquitous, diverse group of molecules present in multiple forms in all organisms. These inhibitors inactivate proteases from predators/pathogens in addition to regulating intracellular proteolysis. In addition to intracellular localization, storage organs of plants are also a potential site of protease inhibitors. Proteins with trypsin inhibitory activity were isolated from Nigella sativa seed extracts by ammonium sulphate precipitation. Extraction conditions were optimized by choosing an optimum solvent, temperature and incubation period. The highest inhibitory activity of protein extracts was achieved by using 50 mM Tris buffer as solvent and overnight incubation of the suspension at 4°C. The crude seed extract fractionated at 60% ammonium sulphate concentration exhibited highest trypsin inhibitory activity, i.e., 60.15 ± 2.95 %, which was comparable to soybean trypsin inhibitor used as positive control. Ammonium sulphate precipitation of crude extract yielded 39.83-fold purification. Partially purified trypsin inhibitor exhibited 2.39±0.23 TIU mg-1. Additionally, Nigella sativa protein extracts were also investigated for their health-promoting effects against two important proteases, α-Dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Highest inhibitory activity against ACE was shown by the crude extract of N. sativa. Among AS fractions, 30% ammonium sulphate concentration exhibited highest inhibition activity against ACE and DPP-IV. Our results suggest that the widely believed role of N. sativa in control of hypertension may at least be partially shared by inhibition of ACE. This is the first study conducted to evaluate the biological activity of N. sativa protein extracts suggesting a potential use of N. sativa proteins in management of hypertension as well as an important source of trypsin inhibitor. Further identification, purification and characterization of different bioactive compounds from N. sativa are being carried out.