The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll-like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single-stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole-cell environment. Here, we engineer the first light-controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.