Abstract Nucleotides, such as uridine 5′‐monophosphate (5′‐UMP) and cytidine 5′‐monophosphate (5′‐CMP), are heavily demanded as food additives, dietary supplements, and medicinal intermediates. Although enzymatic phosphorylation is an effective approach to producing these nucleosides, its practical application is challenging due to the need for the expensive enzymes and coenzyme guanosine‐5′‐triphosphate (GTP). Here we prepared biocompatible calcium‐based nanoflowers incorporated with cascade enzymes, uridine‐cytidine kinase (UCK) and acetate kinase (ACK), for the production of nucleotides. The spatial distribution of these enzymes was optimized to maximize their catalytic performances. The most active nanoflowers (Ca3(PO4)2& ACK)& UCK that display the rate‐limiting enzyme UCK on the outer layer were used in a packed‐bed reactor for continuous synthesis of 5′‐UMP and 5′‐CMP. The catalytic performance of the catalyst retained over 80% within 10 h, showing good operational stability.
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