In the present work, we have developed a new protocol for the purification of chymotrypsin using adsorption onto non-soluble alginate–carrageenan bed. In the first instance, an experimental statistical design was used to screen which parameters are significant in the adsorption process. The variables tested were pH, temperature, and medium’s ionic strength. Significant statistical differences (p < 0.05) were obtained for pH and the medium’s ionic strength, whereas temperature had no influence on the adsorption efficiency in the studied range. In a second step, response surface methodology was used to find the conditions that produced the highest adsorption of chymotrypsin. The optimal working conditions found were pH 6.5 without the addition of salts, which is consistent with an electrostatic interaction. The adsorption kinetics were studied, showing that the adsorption of the enzyme followed a pseudo-first-order rate expression. Different adsorption isotherm models were applied to describe the equilibrium isotherms. The Freundlich model was found to be more suitable for our data. At optimal conditions, 1 g of hydrated matrix adsorbed 11 mg of chymotrypsin. In the desorption process, 93.1% of the biological activity of chymotrypsin was recovered in only 20 min. Additionally, experimental breakthrough profiles were acquired. The optimized conditions were applied in the design of both batch and packed bed adsorption systems for the purification of chymotrypsin from fresh bovine pancreas, reaching a purification factor of 3.8 and 6.9, respectively.