The immobilization of Bacillus psychrosaccharolyticus nucleoside 2′-deoxyribosyltransferase was deeply investigated and finally optimized. The best immobilization procedure resulted to be ionic adsorption on PEI 600 Da agarose followed by crosslinking with 70% oxidized dextran (20 kDa). The percentage of recovered activity was further improved (from 21% to 33%) by the addition of 20% glycerol to the immobilization mixture. The resulting biocatalyst showed a stability profile similar to that of the soluble enzyme and it was used for the preparative synthesis of trifluridine and decytabine obtaining conversions ranging from 50% to 76%. ; Comunidad Autónoma de Madrid (Spain) (Grant S2009/PPQ-1752) and Spanish Ministry of Science and Innovation (CTQ2009-11543) ; 4.312 JCR (2015) Q1, 4/71 Chemistry, applied, 34/144 Chemistry, physical, 13/135 Engineering, chemical ; UEM