<p>Riboswitches are conserved RNA elements that specifically recognize the cognate metabolites and regulate downstream gene expression involved in the metabolic pathways. To date, two classes of xanthine-responsive riboswitches involved in xanthine homeostasis have been identified. The recently reported xanthine-II riboswitch originates from guanine riboswitch family, featuring a single U-to-G mutation and several nucleotide insertions. Here, we report the complex structure of xanthine-II riboswitch bound to xanthine. The tertiary structure of xanthine-II riboswitch adopts a three-way junction scaffold similar to that of guanine riboswitch. However, the distinctive mutation and insertions in xanthine-II riboswitch facilitate the formation of a highly specific binding pocket for xanthine, distinguishing it from guanine riboswitches. Xanthine is bound in the junction region, forming a base triple with C64 and the mutant nucleotide G37, and is sandwiched by one base pair U8-A38 and one base triple A7-C36-U65. Structural alignment and ligand recognition specificity of the xanthine-II riboswitch are further verified by ligand-binding assays of structure-based mutation using isothermal titration calorimetry. Furthermore, leveraging the ligand specificity of the xanthine-II riboswitch, we develop a highly specific and sensitive biosensor for xanthine detection by fusing xanthine-II riboswitch with Pepper fluorogenic aptamer, highlighting the potential applications of xanthine-II riboswitch in diagnosing diseases related to xanthine metabolism disorders.</p>