Trypsin inhibitor from the root-tuber of underutilized legume Winged bean (Psophocarpus tetragonolobus (L.) DC.) (WbT-TI) was purified using ion exchange chromatography followed by size-exclusion chromatography. The purified WbT-TI showed a molecular mass of 20,609 Da and an isoelectric point of 5.10. Ultraviolet circular dichroism (UV-CD) and intrinsic fluorescence reported, that WbT-TI interacts with trypsin. Domain-wise analysis of WbT-TI revealed it to belong to the Kunitz-type soybean trypsin inhibitor (STI) family with a specific β-trefoil fold. The sequence of WbT-TI showed 44% sequence coverage to acidic trypsin inhibitor from the seed of the same plant. Protein interaction similarity analysis (PIPSA) evaluated the electrostatic properties of WbT-TI and provided information about the interacting partners of trypsin inhibitors. The purified protein was quantified and tested for in vitro anticancer activity using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay against the human osteosarcoma (MG-63) cell line. At 5 µg/ml of WbT-TI, the highest inhibition was seen. These studies may lead to the development of winged bean protease inhibitor-based preventive and therapeutic strategies for different kinds of cancers.