The aim of this study consists in the preparation of biocatalysts prepared by immobilizing a proteolytic enzyme (trypsin) via multipoint covalent attachment on agarose beads prepared with different polymer concentration (4% m/m – 4BCL, 6% m/m – 6BCL and 10% m/m – 10BCL). These gels were previously activated with glycidol to produce glyoxyl-agarose beads with different density of active groups. The effect of support type on the catalytic properties of the biocatalysts prepared was assayed in the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPNA). Activated supports achieved 46, 89 and 93 µmols of glyoxyl groups/ml gel using 4BCL, 6BCL and 10 BCL, respectively. The enzyme was fully immobilized on the different gels by offering protein loading of 2 mg/g of support. The hydrolytic activity of the biocatalysts prepared were 1510.0 (4BCL), 993.3 (6BCL) and 411.1 (10BCL) UA/g of support. Conversely, a decrease of recovered activity by increasing the concentration of polymer in their composition was observed (34.3% for 4BCL, 27.7% for 6 BCL and 7.4% for 10BCL). These results could be attributed to diffusional limitation effects of the substrate molecules to the internal microenvironment of the biocatalyst due to possible average porous size reduction by increasing the concentration of polymer. The effect of reaction on the catalytic activity of the biocatalysts was also studied in the hydrolysis of BAPNA at pH 8.0. Soluble trypsin exhibited maximum hydrolytic activity at 50°C, whereas the immobilized enzyme on the three different gels was more active around 75-85°C. These results indicate a possible rigidification of the three-dimensional structure of the enzyme after immobilization on activated agarose beads. The protocol used showed to be promising in the preparation of highly active and thermal-stable biocatalysts for food industries.