The aim of the present work was to purify cysteine protease inhibitors from rapeseed (Brassica napus L.), with potential activity on digestive protease of Colorado Potato Beetle (CPB), Leptinotarsa decemlineata (Say). Ammonium sulfate precipitated proteinaceous fractions; 30, 50, 70, and 100% showed 39.07, 57.03, 51.47, and 22.44% inhibition on the fourth instar larval gut general protease activity, respectively. Fraction 50% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. Gel assays approved the inhibition of the enzyme activity. Fraction 50% was purified by using various chromatography techniques; ion-exchange using DEAE, gel filtration and affinity using SiO2-CPB larval gut homogenate. Three peaks of protein were eluted from ion exchange chromatography using NaCl step gradient, also from gel filtration chromatography. When Z-Ala-Arg-Arg-4mBNA was used as cysteine protease substrate, the purification fold of second fraction of ion exchange chromatography was obtained 24.80, also the yield was 59.09%, the third fraction of gel permeation resulted in a 25.60 fold purification with 28.53% of recovery, and the fraction of affinity chromatography obtained a 22.72 fold purification and yielded 36.35%. In the SDS-PAGE, apparent molecular mass of purified proteins were 34 and 32 kDa by ion-exchange and 24 and 22 kDa by affinity. However, gel filtration was not an appropriate method in this study, because the purified protein band(s) were not observed on the gel. Consequently, these chromatography methods were appropriate methods to purification of inhibitor cystatins, specially affinity which was prepared by using CPB gut enzyme as ligand and obtained specific inhibitor proteins of CPB gut protease activity.