The production of xylooligosaccharides (XOS) was evaluated using immobilized and stabilized biocatalysts of a commercial enzymatic cocktail, Bioxilanase L PLUS (BIO), which is based on the xylanolytic enzymes produced by Trichoderma reesei. BIO was immobilized by multipoint covalent attachment under alkaline conditions on agarose beads highly activated with aldehyde groups (Ag-G BIO) resulting in a highly active and stable biocatalyst (half-life was approximately 50 h at pH 7.0 and 60 degrees C). Ag-G BIO was 10-fold more stable than soluble preparation at pH 7.0 and 60 degrees C. Ag-G BIO was also physically modified by surface coating with polyethyleneimine (PEI) which promotes an ionic interaction with the anionic groups of the enzyme surface. Ag-G BIO covered with a layer of PEI 10 kDa (Ag-G BIO-PEI 10) was > 100-fold more stable than soluble BIO preparation. The optimal biocatalyst (Ag-G BIO-PEI 10) allowed to perform ten cycles of beechwood xylan hydrolysis reaction at high concentration (4% (w/v)) with a high conversion degree (> 80%). Moreover, Ag-G BIO-PEI 10 reached 90% of conversion in only 8 h and so, it could be used for short reaction times, which would extend its useful life, thus allowing its application for industrial processes.