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%.