Fusarium oxysporum f.sp. ciceris (Foc) is one of the most important fungal pathogens of chickpea and is regarded as a constant threat in tropical and subtropical countries. In order to correlate Fusarium wilt resistance/susceptibility in Cicer arietinum to the presence or absence of trypsin inhibitor (TI) in the crude extract, trypsin inhibitory assay (TIA) and in vitro activity of TI against Foc were studied. In the present study, a 20 kDa trypsin inhibitor was purified from Fusarium wilt resistant cultivar (viz. JG 2001-12) by ammonium sulfate precipitation, dialysis and chromatographies with Sephadex G-100 and Diethyl aminoethyl cellulose (DEAE-cellulose-52) ion-exchange column. Results of pathogenecity assay were found to be in correlation to the trypsin inhibitor assay where the Fusarium wilt resistant cultivar showed high trypsin inhibitory activity (99%) in the presence of trypsin enzyme using both natural and synthetic substrates. Preliminary studies using crude extracts of JG 2001-12 showed a decrease in radial growth of Foc. A 45%-82% reduction in conidium germination at 20 μg·mL-1 Cicer arietinum trypsin inhibitor (CaTI) concentration was observed, thereby, indicating the use of CaTI in suppression of pathogen and in its deployment through transgenic plants for the management of Fusarium wilt.