Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.