Chitooligosaccharides (COS) show interesting bioactivities, being a promising product for the pharmaceutical industry. The use of immobilized enzymes for the COS is interesting, as long as the enzyme is stable for industrial purposes. This paper investigated the best condition for immobilization of chitosanase, cellulase and β-glucosidase via adsorption and covalent bond aiming chitosan hydrolysis. For both strategies, the conditions were varied to define the best method for each enzyme, and then they were compared in stability tests. The results showed the optimum hydrolysis temperature for all enzymes was 55 °C, with pH 6.0 for chitosanase and cellulase and pH 4.0 for β-glucosidase. In relation to the immobilization by adsorption, the pH 5.0 was the best for all enzymes, and the enzyme load of 25 U/gsupport was the best for cellulase and chitosanase, while 50 U/gsupport was the best for β-glucosidase. Considering the immobilization by covalent bonding, the optimum conditions depend on the enzyme. For cellulase best result was using 25 U/gsupport and 0.5% (v/v) of glutaraldehyde. In relation to β-glucosidase it was 25 U/gsupport and 1.0% (v/v) of glutaraldehyde and for chitosanase was 50 U/gsupport and 0.5% (v/v) of glutaraldehyde. Immobilization steps improved the thermal stability of all enzymes. In addition, compared to β-glucosidase and cellulase, the chitosanase immobilized via adsorption showed a high operational stability, retaining 70% of the initial enzymatic activity after 6 cycles of hydrolysis. Thus, simple immobilization techniques prove to be a feasible methodology to be implemented in the chitooligosaccharides production2023-10