Abstract Deoxyadenosine kinase from Dictyostelium discoideum ( Dd dAK) phosphorylates its natural substrate (2’‐deoxyadenosine, dAdo) as well as the arabinosyladenine analogues vidarabine (araA) and fludarabine (F‐araA) to their corresponding 5’‐monophosphates. Dd dAK has been here immobilized by ionic interaction on an aminated epoxy‐functionalized support (Sepabeads TM EC‐EP), and cross‐linked with oxidized dextran. The final activity recovery was 33–42 %, depending on the protein loading. Immobilization enhanced the stability of Dd dAK at pH 10 and, to a lesser extent, at 45 °C. Phosphorylation of dAdo, araA and F‐araA catalyzed by immobilized Dd dAK was always nearly quantitative in less than 12 hours. Fludarabine monophosphate was synthesized from F‐araA (95 % conversion, 20 g/L) using immobilized Dd dAK in fully aqueous medium, thus showing that this biocatalyst could be used for developing a preparative phosphorylation process greener and more efficient than POCl 3 ‐based phosphorylation.