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This study aimed to encapsulate natural killer (NK) cells in a hydrogel to sustain their function within the hypoxic tumour microenvironments. An alginate-gelatine hydrogel was generated via electrospray technology. Hydrogel biocompatibility was assessed through cell counting kit-8 and Live/Dead assays to ascertain cell. Moreover, we analysed lactate dehydrogenase assays to evaluate the cytotoxicity against tumours and utilised RT-qPCR to analyse cytokine gene level. Alginate and gelatine formed hydrogels with diameters ranging from 489.2 ± 23.0 μm, and the encapsulation efficiency was 34.07 ± 1.76%. Encapsulated NK cells exhibited robust proliferation and tumour-killing capabilities under normoxia and hypoxia. Furthermore, encapsulation provided a protective shield against cell viability under hypoxia. Importantly, tumour-killing cytotoxicity through cytokines upregulation such as granzyme B and interferon-gamma was preserved under hypoxia. The encapsulation of NK cells not only safeguards their viability but also reinforces anticancer capacity, countering the inhibition of activation induced by hypoxia. [ABSTRACT FROM AUTHOR]