Enzymes are versatile catalysts for numerous industrial biocatalytic processes. Cross-linked enzyme aggregates (CLEAs) as a carrier free immobilization approach has drawn much attention being simple, cost efficient, capable of preserving high catalytic efficiency and improve enzyme reusability. The aim of this study was to develop a reusable, thermally and operationally stable trypsin CLEAs through co-aggregation with chitosan (CHS). Physicochemical characterization of the prepared CLEAs, including pH and temperature optimum, kinetic parameters, and operational and thermal stability in the absence (CLEA-T), and presence (CLEA-T-CHS) of CHS was carried out. CLEA-T-CHS and CLEA-T were prepared under mild conditions and cross linked using glutaraldehyde with 92% and 31% residual activity, respectively. Immobilized trypsin showed improved pH stability at alkaline pH. At 70EC the immobilized enzyme had 62% residual activity while the free enzyme lost 91% of its initial activity. The kinetic parameters (Km and Vmax) of the immobilized trypsin marginally increased, leading to a decreased catalytic efficiency. Operational and thermal stability were highly improved for CLEA-T-CHS; the half-life (t 1/2) of free trypsin and CLEA-T-CHS were 15 min and 65 min, respectively. Storage stability was highly improved; CLEAT-CHS and the free enzyme had 82% and 21% residual activity, respectively, after storage for 4 weeks. CLEA-TCHS retained 64% residual activity after five consecutive hydrolytic cycles, thus reinforcing its robust potentials. In this study, we successfully prepared a thermally stable and highly active immobilized trypsin through crosslinking in the presence of CHS. Results suggest that CLEA-T-CHS has great potential for industrial applications, including re-use in protein digestion.