β-Xylosidases have important applications in many biotechnological processes. In this context, the aim of this work was the purification, immobilization and characterization of a β-xylosidase produced by a new isolate of Aspergillus niger USP-67. β-Xylosidase was produced on static conditions in liquid Benassi medium supplemented with xylan birchwood, initial pH 3.0, for 6 days, at 30 °C. The enzyme was purified on DEAE-Sepharose followed of Superdex™ 200, and the molecular mass of the β-xylosidase was estimated to be 100 kDa, with 90% similarly to the β-xylosidase xlnD from A. niger (gi 146230215 accession), using MS sequencing. The enzyme was immobilized on DEAE-Sepharose, Polyethyleneimine (PEI)-Sepharose, Q-Sepharose, CM-Sepharose, Sulphopropil-Sepharose and MANAE-agarose, but the best result was obtained with PEI-Sepharose, which presented 94% of immobilization yield. Moreover, this derivative was more thermal stable than the soluble enzyme and other supports, which presented a half-life of about 50 min, at 65 °C. The enzyme immobilized on PEI-Sepharose had an optimum pH more acidic (around 4.5) than the purified enzyme (pH 5.5). Metal ions inhibited the soluble enzyme activity more than the immobilized form; however, Zn2+ increased the activity of the immobilized enzyme in 29%. The specific activity of the immobilized enzyme corresponded to 98.15 U/mg, but the soluble enzyme was 77.96 U/mg. Furthermore, the KM and Kcat values for the purified enzyme with p-nitrophenyl-xylopyranoside as substrate were 0.654 mM and 58.87 s−1 and for the immobilized enzyme the values were 0.587 mM and 88.95 s−1, respectively. The purified enzyme efficiently hydrolyzed xylooligosaccharides until xylose, but other xylooligosaccharides (X2–X6) were formed, suggesting transxylosylation action. The immobilized β-xylosidase of A. niger was not inhibited by xylose (100 mM) and glucose (200 mM), what confers to this enzyme a potential application in biotechnological processes.