Photochemical control of oligonucleotides bears great potential for the spatio-temporal control of therapeutic targets, such as immune sensing receptors. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic receptor of the innate immune system that triggers antiviral responses upon detection of viral RNA. RIG-I can be specifically activated by short double-stranded (ds) RNA with a blunt 5' end bearing a triphosphate, mimicking nascent viral transcripts. Tumor cells are specifically sensitive to RIG-I-induced cell death. Here we developed a potent oligonucleotide ligand for spatiotemporally controlled activation of RIG-I by light exposure. Through structural considerations and functional studies we identified a combination of two nucleoside positions in a RIG-I oligonucleotide ligand for which the substitution of both respective 2'-hydroxy groups of the ribose by photolabile protecting groups (2'-photocages) resulted in a complete loss of RIG-I ligand activity, whereas photocaging the individual positions was not sufficient to turn off RIG-I. Light exposure fully restored RIG-I activation by the photocaged RIG-I ligand, enabling light-controlled RIG-I-mediated cell death of human cancer cells which had internalized the photocaged RIG-I ligand prior to light exposure. This novel photoactivatable RIG-I oligonucleotide ligand may be applicable for precise light-controlled induction of tumor cell death in superficial cancer such as melanoma.