An endo-xylanase produced by Aspergillus tamarii Kita was purified using the support CM-cellulose. The enzyme was immobilized on CM-cellulose, glyoxyl-agarose and CNBr-agarose, but the best support was considered the glyoxyl-agarose since its derivative had more stability than the CNBr-agarose derivative (control) in broad range of temperature. The derivatives had an important hyperactivation on glyoxyl-agarose (2.4-fold) and CM-cellulose (5.1-fold). The immobilization of the enzyme improved the physicochemical parameters, since increased enzyme optimum temperature in 5 degrees, which changed from 60 degrees C in the control to 65 degrees C after multipoint covalent immobilization. Thermostability of the glyoxyl-agarose derivative showed half-life (t(0.5)) values of 96 and 60min at 70 and 80 degrees C, respectively. Both derivatives showed apparent optimum pH of 5.5. Glyoxyl-agarose derivative was used in 5 consecutive cycles, maintaining more than 80% of its residual activity. The immobilization of endoxylanase showed to be a very useful method for improving thermostability up to 80 degrees C and enabled to successfully produce xylooligosaccharides (XOS) of 2-5 degree of polymerization (DP 2-5), especially xylotriose (39.7%) and xylotetraose (29.4%), at high temperatures. The glyoxyl-agarose derivative is a potential resource to numerous biotechnological processes, especially in large scale production of XOS which could be used as prebiotic supplementation. (C) 2016 Elsevier Ltd. All rights reserved.