RNA serves as a distinctive pathogen-associated molecular pattern (PAMP) that plays a critical role in innate immunity. However, the specific mechanisms of RNA recognition remain largely unexplored, especially given RNA's vulnerability to degradation and the absence of sequence specificity in most RNA recognition receptors. Notably, Toll-like receptor 13 (TLR13) is capable of detecting a conserved RNA sequence, RNA15 (2054-2068, ACG GAA AGA CCC CGU), within bacterial 23S rRNA, thereby triggering an immune response. To unravel the exact mechanism by which TLR13 recognizes RNA15, we combined experimental approaches with molecular dynamics simulations. Our results suggest that RNA15 adopts a stable hairpin structure in solution, protected from nuclease degradation by intramolecular interactions. TLR13 specifically recognizes this hairpin structure, leading to the dimerization of TLR13. This interaction further induces RNA15 to transition into a stem-loop-like conformation, thereby activating TLR13 downstream signaling. Additionally, our study indicates that TLR13 can form stable dimers in the membrane independently of ligand binding. Although the hairpin structure is the predominant form of RNA15 in solution, the temporary stem-loop-like structure can spontaneously bind to dimeric TLR13, initiating the immune response. These insights deepen our understanding of the complex recognition process of RNA15 by TLR13 and explore the complicated mechanisms governing innate immune system function.