Ligand binding and temperature play important roles in riboswitch RNAs' structures and functions. However, most studies focused on studying structural dynamics or gene-regulation function of riboswitches from the aspect of ligand, instead of temperature. Here we combined NMR, ITC, stopped-flow and assays to investigate the ligand-triggered switch of adenine riboswitch from 10 to 45°C. Our results demonstrated that at single-nucleotide resolution, structural regions sensed ligand and temperature diversely. Temperature had opposite effects on ligand-binding and gene-regulation of adenine riboswitch. Compared with higher temperature, the RNA bound with its cognate ligand obviously stronger, while its regulatory capacity was weakened at lower temperature. In addition, application of specific-labelled RNAs to the stopped-flow experiments identified the real-time folding of the specific positions upon ligand addition at different temperatures. The kissing loop and internal loop at the riboswitch responded to ligand and temperature differently. The distinct thermo-dynamics of adenine riboswitch exposed here may contribute to the fields of RNA sensors and drug design.